Multivalent pneumococcal polysaccharide-protein conjugate composition

ABSTRACT

Provided are mixed carrier, multivalent pneumococcal conjugate compositions comprising 21 different pneumococcal capsular polysaccharide-protein conjugates, wherein each of the conjugates includes a capsular polysaccharide from a different serotype of  Streptococcus pneumoniae  conjugated to either tetanus toxoid (TT) or CRM 197 , wherein the  Streptococcus pneumoniae  serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, where the capsular polysaccharides of serotypes two of serotypes 1, 3, and 5 are conjugated to TT and the remaining capsular polysaccharides are conjugated to CRM 197 . Also provided are methods of producing the mixed carrier, multivalent pneumococcal conjugate compositions and methods of using the same for prophylaxis against  Streptococcus pneumoniae  infection or disease in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and relies on the filing dateof, U.S. provisional patent application No. 62/626,482, filed 5 Feb.2018, and Korean patent application number 10-2018-0045246, filed 18Apr. 2018, the entire disclosures of which are herein incorporated byreference.

TECHNICAL FIELD

This application relates generally to mixed carrier, multivalentpneumococcal conjugate compositions, vaccines comprising the same andmethods of using these compositions and vaccines for prophylaxis ofStreptococcus pneumoniae infection or disease in a subject.

BACKGROUND

Pneumococcus (Streptococcus pneumoniae) is a Gram-positive,lancet-shaped, facultative anaerobic bacteria with over 90 knownserotypes. Most S. pneumoniae serotypes have been shown to causedisease, with the 23 most common serotypes accounting for approximately90% of invasive disease worldwide. Serotypes are classified based on theserological response of the capsular polysaccharides, the most importantvirulence factor for pneumococcus. Capsular polysaccharides are T-cellindependent antigens that induce antibody production in the absence of Thelper cells. T-cell independent antigens generally induce antibodieswith low affinity and short-lived immune responses with little to noimmunological memory.

Initial pneumococcal vaccines included combinations of capsularpolysaccharides from different serotypes. These vaccines can conferimmunity against S. pneumoniae in patients with developed or healthyimmune systems, however, they were not effective in infants, who lack adeveloped immune system, and elderly subjects, who often have impairedimmune function. To improve the immune response to pneumococcalvaccines, particularly in infants and elderly subjects, who are athigher risk to develop S. pneumoniae infection, capsular polysaccharideswere conjugated to suitable carrier proteins to create pneumococcalconjugate vaccines. Conjugation to a suitable carrier protein changesthe capsular polysaccharide from a T-cell independent antigen to aT-cell dependent antigen. As such, the immune response against theconjugated capsular polysaccharide involves T helper cells, which helpinduce a more potent and rapid immune response upon re-exposure to thecapsular polysaccharide.

There are at least two approaches to developing pneumococcal conjugatevaccines: the single carrier approach and the mixed carrier approach.The immunogenicity of different capsular polysaccharide conjugates mayvary depending on the pneumococcal serotype and carrier protein used. Inthe single carrier approach, the capsular polysaccharides from differentserotypes are conjugated to a single protein carrier. Pfizer's PREVNARseries of vaccines is an example of a single carrier approach where thedifferent capsular polysaccharides are conjugated to the CRM₁₉₇ proteincarrier, a non-toxic variant of the diphtheria toxoid having a singleamino acid substitution of glutamic acid for glycine. The 7-valentPREVNAR vaccine (PREVNAR) was first approved in 2000 and contains thecapsular polysaccharides from the seven most prevalent serotypes: 4, 6B,9V, 14, 18C, 19F and 23F. The 13-valent vaccine, PREVNAR 13, added theserotypes 1, 5, 7F, 3, 6A, and 19A to the CRM₁₉₇ protein carrier. Theprotein carrier, CRM₁₉₇, the single carrier used in PREVNAR vaccines hasnever been used as part of a mixed carrier system in a pneumococcalconjugate vaccine.

The second pneumococcal vaccine approach is the mixed carrier approach.In the mixed carrier approach, instead of using a single proteincarrier, two or more protein carriers are used, with capsularpolysaccharides from specific serotypes conjugated to a first proteincarrier and capsular polysaccharides from different serotypes conjugatedto at least a second, different protein carrier. For example,GlaxoSmithKline has developed SYNFLORIX, a 10-valent (serotypes 1, 4, 5,6B, 7F, 9V, 14, 18C, 19F and 23F), mixed carrier, pneumococcal conjugatevaccine that uses H influenzae protein D, tetanus toxoid, and diphtheriatoxoid as the protein carriers. In SYNFLORIX, serotypes 1, 4, 5, 6B, 7F,9V, 14, and 23F are conjugated to protein D; serotype 18C is conjugatedto tetanus toxoid; and serotype 19F is conjugated to diphtheria toxoid[2]. Serotype 3 was removed from the 11-valent precursor to SYNFLORIX,in part, because it did not show serotype-specific efficacy in an acuteotitis media trial [1]. Another group, Aventis Pasteur, developed an11-valent (serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F),mixed carrier, pneumococcal conjugate vaccine using diphtheria toxoidand tetanus toxoid as protein carriers [3]. Capsular polysaccharidesfrom serotypes 3, 9V, 14, and 18C can evoke a better response whenconjugated to diphtheria toxoid than they do when conjugated to tetanustoxoid [6]. Thus, serotypes 3, 6B, 14, and 18C were conjugated todiphtheria toxin and serotypes 1, 4, 5, 7F, 9V, 19F, and 23F wereconjugated to tetanus toxoid. The development of this mixed carrier,pneumococcal vaccine was terminated due, in part, to technical reasonsand the potential of a reduced response when administered with acellularpertussis vaccines [3]. Recently, serotype 5 as well as 1 was reportedas having one of the lowest observed OPA titers from all PREVNAR 13serotypes, in which there was an associated correlation between IgGtiter and OPA activity [4]. Also it was suggested that for serotype 3, amuch higher serum IgG concentration would be needed for protection [5].

SUMMARY

This application provides new and improved mixed carrier, multivalentpneumococcal conjugate compositions and vaccines comprising the same. Inone aspect, this application provides a mixed carrier, multivalentpneumococcal conjugate composition, comprising 21 different pneumococcalcapsular polysaccharide-protein conjugates, wherein each pneumococcalcapsular polysaccharide-protein conjugate comprises a protein carrierconjugated to a capsular polysaccharide from a different serotype ofStreptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypesare selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the protein carrier isCRM₁₉₇ or tetanus toxoid, wherein two of the capsular polysaccharidesare conjugated to tetanus toxoid and the remaining capsularpolysaccharides are conjugated to CRM₁₉₇, and wherein the two capsularpolysaccharides that are conjugated to tetanus toxoid are selected fromthe group consisting of serotypes 1, 3, and 5.

In some embodiments of the mixed carrier, 21-valent pneumococcalconjugate composition, the capsular polysaccharides from serotypes 1 and5 are conjugated to tetanus toxoid, and the capsular polysaccharidesfrom serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇.

In another embodiment of the mixed carrier, 21-valent pneumococcalconjugate composition, the capsular polysaccharides from serotypes 1 and3 are conjugated to tetanus toxoid, and the capsular polysaccharidesfrom serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇.

In another embodiment of the mixed carrier, 21-valent pneumococcalconjugate composition, the capsular polysaccharides from serotypes 3 and5 are conjugated to tetanus toxoid, and the capsular polysaccharidesfrom serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇.

In some embodiments, the mixed carrier, multivalent pneumococcalconjugate composition further comprises an adjuvant, such as analuminum-based adjuvant, including, but not limited to aluminumphosphate, aluminum sulfate, and aluminum hydroxide.

Another aspect is directed to the use of the mixed carrier, 21-valentpneumococcal conjugate composition as a vaccine.

Yet another aspect is directed to a vaccine comprising the mixedcarrier, 21-valent pneumococcal conjugate composition and apharmaceutically acceptable excipient.

Yet another aspect is directed to a method for prophylaxis ofStreptococcus pneumoniae infection or disease in a subject, such as ahuman, the method comprising administering a prophylactically effectiveamount of the mixed carrier, 21-valent pneumococcal conjugatecompositions or a vaccine comprising the same to the subject.

In certain embodiments, the subject is a human who is at least 50 yearsold and the disease is pneumonia or invasive pneumococcal disease (IPD).

In other embodiments, the subject is a human who is at least 6 weeks oldand the disease is pneumonia, invasive pneumococcal disease (IPD), oracute otitis media (AOM). In some embodiments, the human subject is 6weeks to 5 years of age. In other embodiments, the human subject is 2 to15 months of age or 6 to 17 years of age.

In certain embodiments, the mixed carrier, 21-valent pneumococcalconjugate composition or vaccine is administered by intramuscularinjection. In certain embodiments, the mixed carrier, 21-valentpneumococcal conjugate composition or vaccine is administered as part ofan immunization series.

Yet another aspect is directed to an immunogenic conjugate ofStreptococcus pneumoniae serotype 9N, which contains: a serotype 9Ncapsular saccharide from Streptococcus pneumoniae; and a carrier proteinbound to the capsular saccharide, wherein the carrier protein is CRM₁₉₇.In certain embodiments of the immunogenic serotype 9N conjugate, themixed carrier, multivalent pneumococcal conjugate compositions andvaccines (and methods/uses of the same), the serotype 9N saccharide maybe bound to CRM₁₉₇ to form a conjugate in a state where it is activatedto have a degree of oxidation of 2-19 or 5-10 and a molecular weight of200-700 kDa. In certain embodiments of the immunogenic serotype 9Nconjugate, the mixed carrier, multivalent pneumococcal conjugatecompositions and vaccines (and methods/uses of the same), theimmunogenic serotype 9N conjugate may have a molecular weight of500-4,000 kDa.

In certain embodiments of the immunogenic serotype 9N conjugate, themixed carrier, multivalent pneumococcal conjugate compositions andvaccines (and methods/uses of the same), the ratio of the serotype 9Ncapsular saccharide to the carrier protein in the immunogenic serotype9N conjugate is 0.1-5 (w/w). In certain embodiments, the ratio is0.5-2.5.

In certain embodiments of the immunogenic serotype 9N conjugate, themixed carrier, multivalent pneumococcal conjugate compositions andvaccines (and methods/uses of the same), 15-60% of the immunogenicserotype 9N conjugate may have a K_(d) of 0.3 or below in a CL-4Bcolumn.

In certain embodiments of the immunogenic serotype 9N conjugate, themixed carrier, multivalent pneumococcal conjugate compositions andvaccines (and methods/uses of the same), the immunogenic serotype 9Nconjugate has been prepared with a serotype 9N polysaccharide that hasbeen activated to achieve a degree of oxidation of 2-19. In certainembodiments, the immunogenic serotype 9N conjugate has been preparedwith a serotype 9N polysaccharide that has been activated to achieve adegree of oxidation of 5-10.

In certain embodiments of the immunogenic serotype 9N conjugate, themixed carrier, multivalent pneumococcal conjugate compositions andvaccines (and methods/uses of the same), when the Streptococcuspneumoniae serotype 9N saccharide is conjugated with the CRM₁₉₇ byadding 0.02-0.19 μg of periodate per 1 μg of sugar, the conjugate mayhave a molecular weight of 500-4,000 kDa, a molecular weightdistribution of 15-60% (K_(d)≤0.3) and a saccharide/protein ratio of0.5-2.5.

In yet another aspect, the present disclosure also provides a method forpreparing an immunogenic conjugate of Streptococcus pneumoniae serotype9N, the method comprising:

(a) lysing a bacterial cell producing Streptococcus pneumoniae serotype9N capsular polysaccharide by fermenting the same;

(b) purifying Streptococcus pneumoniae serotype 9N capsular saccharidefrom the lysed cell;

(c) activating the Streptococcus pneumoniae serotype 9N capsularpolysaccharide by reacting with an oxidizing agent to achieve a degreeof oxidation of 2-19 or 5-10; and

(d) forming a conjugate of the Streptococcus pneumoniae serotype 9Ncapsular saccharide bound to CRM₁₉₇ by mixing the activated saccharidewith CRM₁₉₇.

In certain embodiments, the CRM₁₉₇ mixed in the step (d) may be reactedwith a reducing agent to form the conjugate with the activatedStreptococcus pneumoniae serotype 9N capsular polysaccharide. In certainembodiments, in step (c), 0.02-0.19 μg of periodate may be reacted with1 μg of the Streptococcus pneumoniae serotype 9N capsular polysaccharideat 20-25° C. for 15-20 hours.

In certain embodiments, the Streptococcus pneumoniae serotype 9Ncapsular polysaccharide reacted with the oxidizing agent in step (c) mayhave a molecular weight of 400-900 kDa. In certain embodiments, theactivated Streptococcus pneumoniae serotype 9N capsular polysaccharidemixed with the CRM₁₉₇ in step (d) may have a molecular weight of 200-700kDa. In certain embodiments, the immunogenic conjugate of Streptococcuspneumoniae serotype 9N may have a molecular weight of 500-4,000 kDa. Incertain embodiments, an initial input ratio of the CRM₁₉₇ to theactivated serotype 9N capsular saccharide (carrier CRM₁₉₇:saccharide)may be 0.5-2.5:1. In certain embodiments, at least 15-60% of theimmunogenic conjugate may have a K_(d) of 0.3 or below as measured in aCL-4B column.

In certain embodiments, when the Streptococcus pneumoniae serotype 9Npolysaccharide of the present disclosure is conjugated with the CRM₁₉₇by adding 0.02-0.19 μg of periodate per 1 μg of sugar, the immunogenicconjugate has a molecular weight of 500-4,000 kDa, a molecular weightdistribution of 15-60% (K_(d)≤0.3) as measured in a CL-4B column and aCRM₁₉₇/polysaccharide ratio of 0.5-2.5.

The foregoing and other objects, features, and advantages of the mixedcarrier, 21-valent pneumococcal conjugate compositions will become moreapparent from the following detailed description.

Definitions

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms may be set forth through thespecification.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Administer: As used herein, “administering” a composition to a subjectmeans to give, apply or bring the composition into contact with thesubject. Administration can be accomplished by any of a number ofroutes, such as, for example, topical, oral, subcutaneous,intramuscular, intraperitoneal, intravenous, intrathecal andintradermal.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 140%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Conjugate: As used herein, and understood from the proper context, theterms “conjugate(s)” or “glycoconjugate(s)” refer to a Streptococcuspneumoniae polysaccharide conjugated to a carrier protein using anycovalent or non-covalent bioconjugation strategy.

Degree of oxidation: As used herein, the term “degree of oxidation” (DO)refers to the number of sugar repeat units per aldehyde group generatedwhen a purified or sized saccharide is activated with an oxidizingagent. The degree of oxidation of a saccharide can be determined usingroutine methods known to those of ordinary skill in the art.

Excipient: As used herein, the term “excipient” refers to anon-therapeutic agent that may be included in a composition, for exampleto provide or contribute to a desired consistency or stabilizing effect.

Mixed carrier: As used herein, a mixed carrier, pneumococcal conjugatecomposition refers to a pneumococcal conjugate composition having morethan one type of protein carrier.

Mixed carrier, 21-valent pneumococcal conjugate composition: As usedherein, the term “mixed carrier, 21-valent pneumococcal conjugatecomposition(s)” refers to a composition comprising or consisting of 21different pneumococcal capsular polysaccharide-protein conjugates,wherein each pneumococcal capsular polysaccharide-protein conjugatecomprises a protein carrier conjugated to a capsular polysaccharide froma different serotype of Streptococcus pneumoniae, wherein theStreptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N,9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, whereinthe protein carrier is CRM₁₉₇ or tetanus toxoid, wherein two of thecapsular polysaccharides are conjugated to tetanus toxoid and theremaining capsular polysaccharides are conjugated to CRM₁₉₇, and whereinthe two capsular polysaccharides that are conjugated to tetanus toxoidare selected from the group consisting of serotypes 1, 3, and 5. In someembodiments, the capsular polysaccharides from serotypes 1 and 5 areconjugated to tetanus toxoid, and the capsular polysaccharides from theremaining serotypes are conjugated to CRM₁₉₇. In some embodiments, thecapsular polysaccharides from serotypes 1 and 3 are conjugated totetanus toxoid, and the capsular polysaccharides from the remainingserotypes are conjugated to CRM₁₉₇. In another embodiment, the capsularpolysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid,and the remaining capsular polysaccharides are conjugated to CRM₁₉₇.

Molecular weight: Unless specified otherwise, as used herein, the term“molecular weight” of a capsular saccharide or a capsularsaccharide-carrier protein conjugate refers to the average molecularweight calculated by size exclusion chromatography (SEC) in combinationwith multi-angle laser light scattering (MALLS).

Multivalent: As used herein, the term “multivalent” refers to apneumococcal conjugate composition having pneumococcal capsularpolysaccharides from more than one Streptococcus pneumoniae serotype.

Pharmaceutically acceptable excipient: The pharmaceutically acceptableexcipients useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15^(th) Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of one or more therapeuticcompositions, including vaccines, and additional pharmaceutical agents.Suitable pharmaceutical excipients include, for example, starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. In general, the nature of the excipient will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, buffers, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions (forexample, powder, pill, tablet, or capsule forms), conventional non-toxicsolid excipients can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, a surface activeagent, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Prophylactically Effective Amount: As defined herein, the term “aprophylactically effective amount” or “a prophylactically effectivedose” refers to the amount or dose required to induce an immune responsesufficient to delay onset and/or reduce in frequency and/or severity oneor more symptoms caused by an infection with Streptococcus pneumoniae.

Prophylaxis: The term “prophylaxis,” as used herein, refers to avoidanceof disease manifestation, a delay of onset, and/or reduction infrequency and/or severity of one or more symptoms of a particulardisease, disorder or condition (e.g., infection with Streptococcuspneumoniae). In some embodiments, prophylaxis is assessed on apopulation basis such that an agent is considered to provide prophylaxisagainst a particular disease, disorder or condition if a statisticallysignificant decrease in the development, frequency, and/or intensity ofone or more symptoms of the disease, disorder or condition is observedin a population susceptible to the disease, disorder, or condition.

Subject: As used herein, the term “subject” means any mammal, includingmice, rabbits, and humans. In certain embodiments the subject is anadult, an adolescent or an infant. In some embodiments, terms“individual” or “patient” are used and are intended to beinterchangeable with “subject.”

DETAILED DESCRIPTION

The following description of the disclosed embodiment(s) and Examples ismerely exemplary in nature and is in no way intended to limit theinvention, its application, or uses.

This application provides new and improved mixed carrier, multivalentpneumococcal conjugate compositions and vaccines comprising the same.While the protein carrier, CRM₁₉₇, has previously been used in singlecarrier, pneumococcal conjugate vaccines, this application describes theuse of CRM₁₉₇ in a mixed carrier, pneumococcal conjugate vaccine. Inparticular, this application describes the combined use of CRM₁₉₇ andtetanus toxoid as carrier proteins for specific pneumococcal serotypesin multivalent pneumococcal conjugate compositions and vaccines.

As discussed above, the immunogenicity of different capsularpolysaccharide conjugates may vary depending on the pneumococcalserotype and carrier protein used. This application describes thesuccessful conjugation of serotype 3 to tetanus toxoid as part of amixed carrier vaccine, notwithstanding previous teachings that serotype3 was more immunogenic when conjugated to diphtheria toxoid rather thantetanus toxoid [6]. It also describes the successful conjugation ofserotypes 1 and 5 to tetanus toxoid as part of a mixed carrier vaccine.It also discloses the unexpected finding that the antibody response toserotype 3 conjugated to tetanus toxoid in a mixed carrier,multi-valent, for example 21-valent, pneumococcal conjugate compositionwas about 4-5 fold higher than when serotype 3 was conjugated to CRM₁₉₇in a single carrier, 13-valent pneumococcal conjugate composition(PREVNAR 13).

Further, the unexpected finding was not limited to serotype 3 but wasalso observed for other serotypes conjugated to tetanus toxoid in amixed carrier, multi-valent pneumococcal conjugate composition. As shownin the Examples, conjugation of serotypes 1 and 5 or 3 and 5 to tetanustoxoid in a mixed carrier, pneumococcal conjugate composition with theremaining serotypes conjugated to CRM₁₉₇ (e.g., PCV21(1/5)-TT andPCV21(3/5)-TT) consistently induced significantly enhanced antibodyresponses to the serotypes conjugated to tetanus toxoid as compared tothe antibody responses (IgG response or MOPA titers) against the sameserotypes conjugated to CRM₁₉₇ in a single carrier, pneumococcalconjugate composition (PREVNAR 13).

The mixed carrier, 21-valent pneumococcal conjugate compositionsdescribed in this application also include pneumococcal serotypes notcurrently covered by the three pneumococcal conjugate vaccines currentlyavailable on the global market: PREVNAR (called Prevenar in somecountries), SYNFLORIX and PREVNAR 13. Disease caused by pneumococcalserotypes not currently covered is on the rise, due, in part, to thedevelopment of antibacterial resistance, the increased number ofimmunocompromised patients, and lack of immune pressure. For example,none of the currently available pneumococcal conjugate vaccines includesserotype 9N. In addition, none of the currently available pneumococcalconjugate vaccines includes serotypes 8, 10A, 11A, 12F, 15B, 22F and33F. The present disclosure demonstrates the successful implementationof serotypes 8, 9N, 10A, 11A, 12F, 15B, 22F and 33F into a mixed carrier(tetanus toxoid and CRM₁₉₇), pneumococcal conjugate vaccine, as well asserotype 9N induced antibody responses that were about 40- to 50-foldhigher than PREVNAR13.

Pneumococcal Polysaccharide Serotype 9N

The serotype 9N polysaccharide may be obtained directly from thebacteria by using an isolation procedure known to those of ordinaryskill in the art (including, but not limited to, the methods disclosedin US Patent Application Publication No. 2006/0228380). In addition, thesaccharide can be produced using synthetic protocols.

The serotype 9N Streptococcus pneumoniae strain may be obtained fromestablished culture collections (e.g., the Streptococcal ReferenceLaboratory of the Centers for Disease Control and Prevention (Atlanta,Ga.)) or clinical specimens.

The bacterial cell is typically grown in a medium, such as a soy-basedmedium. Following fermentation of the bacterial cell producingStreptococcus pneumoniae serotype 9N capsular polysaccharide, thebacterial cell is lysed to produce a cell lysate. Then, the serotype 9Npolysaccharide may be isolated from the cell lysate using purificationtechniques known in the art, including centrifugation, depth filtration,precipitation, ultrafiltration, treatment with activated carbon,diafiltration and/or column chromatography (including, but not limitedto, the methods disclosed in US Patent Application Publication No.2006/0228380). The purified serotype 9N capsular polysaccharide may beused for preparation of an immunogenic conjugate. The serotype 9Ncapsular polysaccharide obtained by purification of the serotype 9Npolysaccharide from the Streptococcus pneumoniae lysate and optionallyby sizing of the purified polysaccharide may be characterized bydifferent parameters including, for example, the molecular weight (MW)of the serotype 9N capsular polysaccharide.

In certain embodiments, embodiments, the purified polysaccharidepurified from the Streptococcus pneumoniae serotype 9N beforeconjugation has a molecular weight of 5-5,000 kDa. In certainembodiments, the serotype 9N capsular polysaccharide before conjugationhas a molecular weight of 50-1,000 kDa. In certain embodiments, theserotype 9N capsular polysaccharide before conjugation has a molecularweight of 70-900 kDa. In certain embodiments, the serotype 9N capsularpolysaccharide before conjugation has a molecular weight of 100-800 kDa.In certain embodiments, the purified serotype 9N capsular polysaccharidemay be activated prior to conjugation to have a molecular weight of50-800 kDa, 80-780 kDa, 100-770 kDa, 120-760 kDa, 140-750 kDa, 150-740kDa, 160-730 kDa, 170-735 kDa, 180-720 kDa, 190-710 kDa, 200-700 kDa,220-690 kDa, 240-680 kDa, 260-670 kDa, 270-660 kDa or similar molecularweight ranges. Any whole number within any of the above ranges iscontemplated as an embodiment of the present disclosure.

The activated serotype 9N polysaccharide may be characterized by adegree of oxidation and molecular weight. In certain embodiments, theactivated serotype 9N polysaccharide may have a degree of oxidation of0.5-25, 0.6-23, 0.8-21, 1-20.8, 1.1-20.5, 1.2-20.3, 1.3-20, 1.4-19.5,1.5-19.3, 1.6-19.2, 1.7-19.1 2-19, 3-18, 4-15, or 5-10.

The polysaccharide may become slightly reduced in size during a normalpurification procedure. Additionally, as described in the presentdisclosure, the polysaccharide may be subjected to sizing beforeconjugation. The molecular weight range mentioned above refers to thatof the purified polysaccharide after the final sizing step (e.g., afterpurification, hydrolysis and activation) before conjugation.

Mixed Carrier, Multivalent Pneumococcal Conjugate Compositions andMethods of Making the Same

This disclosure provides a mixed carrier, multivalent pneumococcalconjugate composition comprising or consisting of 21 differentpneumococcal capsular polysaccharide-protein conjugates, wherein eachpneumococcal capsular polysaccharide-protein conjugate comprises aprotein carrier conjugated to a capsular polysaccharide from a differentserotype of Streptococcus pneumoniae, wherein the Streptococcuspneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, wherein the proteincarrier is CRM₁₉₇ or tetanus toxoid, wherein two of the capsularpolysaccharides are conjugated to tetanus toxoid and the remainingcapsular polysaccharides are conjugated to CRM₁₉₇, and wherein the twocapsular polysaccharides that are conjugated to tetanus toxoid areselected from the group consisting of serotypes 1, 3, and 5.

In some embodiments, the capsular polysaccharides from serotypes 1 and 5are conjugated to tetanus toxoid, and the capsular polysaccharides fromthe remaining serotypes are conjugated to CRM₁₉₇. In another embodiment,the capsular polysaccharides from serotypes 1 and 3 are conjugated totetanus toxoid, and the remaining capsular polysaccharides areconjugated to CRM₁₉₇. In another embodiment, the capsularpolysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid,and the remaining capsular polysaccharides are conjugated to CRM₁₉₇.

In a polysaccharide-protein conjugate vaccine, a carrier protein isconjugated to a polysaccharide antigen primarily to help enhance theimmune response (e.g. antibody response) to the polysaccharide antigen.Carrier proteins are preferably proteins that are non-toxic. Carrierproteins should be amenable to conjugation with a pneumococcalpolysaccharide using standard conjugation procedures, as discussed infurther detail below. The carrier proteins used in the mixed carrier,21-valent pneumococcal conjugate compositions are tetanus toxoid (TT)and CRM₁₉₇, each of which has been used in the design of pneumococcalconjugate vaccines but never in the same, mixed carrier vaccine.

CRM₁₉₇ is a non-toxic variant (i.e., toxoid) of diphtheria toxin thatretains the immunologic properties of the wild type diphtheria toxin.CRM₁₉₇ differs from the wild type diphtheria toxin at a single base inthe structural gene, which gives rise to a single amino acidsubstitution from glutamic acid to glycine. CRM₁₉₇ is typically isolatedfrom cultures of Corynebacterium diphtheria strain C7 (0197) grown oncasamino acids and yeast extract-based medium. CRM₁₉₇ may be purifiedthrough ultra-filtration, ammonium sulfate precipitation, andion-exchange chromatography. Alternatively, CRM₁₉₇ can be preparedrecombinantly in accordance with U.S. Pat. No. 5,614,382, which ishereby incorporated by reference in its entirety. CRM₁₉₇ has been usedin the design of pneumococcal conjugate vaccines but never as part of amixed carrier vaccine.

Tetanus toxoid is prepared and used worldwide for large-scaleimmunization against tetanus (or lockjaw) caused by Clostridium tetani.Tetanus toxoid is also used both singly and in combination withdiphtheria and/or pertussis vaccines. The parent protein, tetanus toxin,is generally obtained in cultures of Clostridium tetani. Tetanus toxinis a protein of about 150 kDa and consists of two subunits (about 100kDa and about 50 kDa) linked by a disulfide bond. The toxin is typicallydetoxified with formaldehyde and can be purified from culture filtratesusing known methods, such as ammonium sulfate precipitation (see, e.g.,[7], [8]) or chromatography techniques, as disclosed, for example, in WO1996/025425. Tetanus toxin may also be inactivated by recombinantgenetic means.

Tetanus toxoid has also been used as a carrier protein in othervaccines, including pneumococcal conjugate vaccines. But using tetanustoxin in a mixed carrier, pneumococcal conjugate vaccine in combinationwith CRM₁₉₇ is new. The art also teaches away from conjugating serotype3 to tetanus toxoid in a mixed carrier, pneumococcal conjugate vaccinebecause serotype 3 was shown to be more immunogenic when conjugated todiphtheria toxoid as compared to tetanus toxoid [6].

The pneumococcal capsular polysaccharides used in the compositions andvaccines described herein, including the capsular polysaccharides fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,18C, 19A, 19F, 22F, 23F, and 33F, may be prepared from Streptococcuspneumoniae using any available technique, including standard techniquesknown to one of ordinary skill in the art, including, for example, thosedisclosed in WO 2006/110381, WO 2008/118752, WO 2006/110352, and U.S.Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071,2007/0184072, 2007/0231340, 2008/0102498 and 2008/0286838, all of whichare incorporated by reference in their entireties. For example, eachpneumococcal capsular polysaccharide serotype may be grown in culturemedium (e.g., a soy-based medium). The cells are lysed, and individualpolysaccharides may be purified from the lysate through centrifugation,precipitation, ultra-filtration, and/or column chromatography. Inaddition, the pneumococcal capsular polysaccharide can be produced usingsynthetic protocols.

Capsular polysaccharides of Streptococcus pneumoniae comprise repeatingoligosaccharide units, which may contain up to 8 sugar residues. Acapsular saccharide antigen may be a full length polysaccharide, or itmay be reduced in size (e.g., a single oligosaccharide unit, or ashorter than native length saccharide chain of repeating oligosaccharideunits). The size of capsular polysaccharides may be reduced by variousmethods known in the art, such as acid hydrolysis treatment, hydrogenperoxide treatment, sizing by a high pressure homogenizer, optionallyfollowed by a hydrogen peroxide treatment to generate oligosaccharidefragments, or microfluidization.

The pneumococcal conjugate of each of the serotypes may be prepared byconjugating a capsular polysaccharide of each serotype to a carrierprotein. The different pneumococcal conjugates may be formulated into acomposition, including a single dosage formulation.

To prepare a polysaccharide-protein conjugate, the capsularpolysaccharides prepared from each pneumococcal serotype may bechemically activated so that the capsular polysaccharides may react witha carrier protein. Once activated, each capsular polysaccharide may beseparately conjugated to a carrier protein to form a glycoconjugate. Thechemical activation of the polysaccharides and subsequent conjugation tothe carrier protein may be achieved by conventional methods.

For example, vicinal hydroxyl groups at the end of the capsularpolysaccharides can be oxidized to aldehyde groups by oxidizing agentssuch as periodates (including sodium periodate, potassium periodate, orperiodic acid), as disclosed, for example, in U.S. Pat. Nos. 4,365,170,4,673,574 and 4,902,506, which are hereby incorporated by reference intheir entireties. The periodate randomly oxidizes the vicinal hydroxylgroup of a carbohydrate to form a reactive aldehyde group and causescleavage of a C—C bond. The term “periodate” includes both periodate andperiodic acid. This term also includes both metaperiodate (IO⁴⁻) andorthoperiodate (IO⁶⁵⁻). The term “periodate” also includes various saltsof periodate including sodium periodate and potassium periodate. Incertain embodiments, the polysaccharide may be oxidized in the presenceof sodium metaperiodate.

In certain embodiments, the periodate may be used in an amount of about0.03-0.17 μg per 1 μg of polysaccharide. In certain embodiments, theperiodate may be used in an amount of about 0.025-0.18 μg or about0.02-0.19 μg per 1 μg of polysaccharide. The saccharide may be activatedas desired within the above range. Outside the range, the effect may beunsatisfactory.

Polysaccharides may also be activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Theactivated polysaccharide is then coupled directly or via a spacer orlinker group to an amino group on the carrier protein.

For example, the spacer could be cystamine or cysteamine to give athiolated polysaccharide which could be coupled to the carrier via athioether linkage obtained after reaction with a maleimide-activatedcarrier protein (for example using N-[y-maleimidobutyrloxy]succinimideester (GMBS)) or a haloacetylated carrier protein (for example usingiodoacetimide, N-succinimidyl bromoacetate (SBA; SIB),N-succinimidyl(4-iodoacetyl)aminobenzoate (SlAB),sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB),N-succinimidyl iodoacetate (SIA) or succinimidyl3-[bromoacetamido]proprionate (SBAP)). Preferably, the cyanate ester(optionally made by COAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (AOH) and the amino-derivatized saccharide isconjugated to the carrier protein using carbodiimide (e.g., EDAC or EDC)chemistry via a carboxyl group on the protein carrier. Such conjugatesare described for example in WO 93/15760, WO 95/08348 and WO 96/129094,all of which are hereby incorporated by reference in their entireties.

The conjugation of the activated capsular polysaccharides and thecarrier proteins may be achieved, for example, by reductive amination,as described, for example, in U.S. Patent Appl. Pub. Nos. 2006/0228380,2007/0231340, 2007/0184071 and 2007/0184072, WO 2006/110381, WO2008/079653, and WO 2008/143709, all of which are incorporated byreference in their entireties. For example, the activated capsularpolysaccharides and the carrier protein may be reacted with a reducingagent to form a conjugate. Reducing agents which are suitable includeborohydrides, such as sodium cyanoborohydride, borane-pyridine, sodiumtriacetoxyborohydride, sodium or borohydride, or borohydride exchangeresin. At the end of the reduction reaction, there may be unreactedaldehyde groups remaining in the conjugates. The unreacted aldehydegroups may be capped using a suitable capping agent, such as sodiumborohydride (NaBH₄). In an embodiment, the reduction reaction is carriedout in aqueous solvent. In another embodiment the reaction is carriedout in aprotic solvent. In an embodiment, the reduction reaction iscarried out in DMSO (dimethylsulfoxide) or in DMF (dimethylformamide)solvent. Other possible reducing agents include, but are not limited to,amine-boranes such as pyridine-borane, 2-picoline-borane,2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3,benzylamine-BH3 or 5-ethyl-2-methylpyridine-borane (PEMB).

The activated capsular polysaccharides may be conjugated directly to thecarrier protein or indirectly through the use of a spacer or linker,such as a bifunctional linker. The linker is optionallyheterobifunctional or homobifunctional, having for example a reactiveamino group and a reactive carboxylic acid group, 2 reactive aminogroups or two reactive carboxylic acid groups.

Other suitable techniques for conjugation use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S-NHS, EDC, TSTU, as described, for example, in International PatentApplication Publication No. WO 98/42721, which is incorporated byreference in their entirety. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with 1,1′-carbonyldiimidazole (CDl) (see Bethell et al.(1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J. Chromatogr.218:509-518) followed by reaction with a protein to form a carbamatelinkage. This may involve reduction of the anomeric terminus to aprimary hydroxyl group, optional protection/deprotection of the primaryhydroxyl group, reaction of the primary hydroxyl group with CDI to forma CDI carbamate intermediate and coupling the CDl carbamate intermediatewith an amino group on a protein.

The ratio of polysaccharide to carrier protein for pneumococcalconjugate vaccines is typically in the range 0.3-3.0 (w/w) but can varywith the serotype. The ratio can be determined either by independentmeasurement of the amounts of protein and polysaccharide present, or bymethods that give a direct measure of the ratio known in the art.Methods including ¹H NMR spectroscopy or SEC-HPLC-UV/RI with dualmonitoring (e.g. refractive index and UV, for total material and proteincontent respectively) can profile the saccharide/protein ratio over thesize distribution of conjugates, as well as by SEC-HPLC-MALLS orMALDI-TOF-MS.

The polysaccharide-protein conjugates thus obtained may be purified andenriched by a variety of methods. These methods includeconcentration/diafiltration, column chromatography, and depthfiltration. The purified polysaccharide-protein conjugates are combinedto formulate a mixed carrier, 21-valent pneumococcal conjugatecomposition, which can be used as a vaccine.

Formulation of a vaccine composition can be accomplished usingart-recognized methods. A vaccine composition is formulated to becompatible with its intended route of administration. The individualpneumococcal capsular polysaccharide-protein conjugates can beformulated together with a physiologically acceptable vehicle to preparethe composition. Examples of such vehicles include, but are not limitedto, water, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol) and dextrose solutions.

In some embodiments, the mixed carrier, 21-valent pneumococcal conjugatecomposition further comprises an adjuvant. As used herein, an “adjuvant”refers to a substance or vehicle that non-specifically enhances theimmune response to an antigen. Adjuvants can include a suspension ofminerals (alum, aluminum salts, such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, aluminum hydroxy phosphate sulfate, etc.)on which antigen is adsorbed; or water-in-oil emulsion in which antigensolution is emulsified in mineral oil (for example, Freund's incompleteadjuvant), sometimes with the inclusion of killed mycobacteria (Freund'scomplete adjuvant) to further enhance antigenicity. Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants (for example, see U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants also include biological molecules, such as lipids andcostimulatory molecules. Exemplary biological adjuvants include AS04[9], IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L and 41 BBL.

In some embodiments, the adjuvant is an aluminum-based adjuvant.Typically, a single 0.5 ml vaccine dose is formulated to contain about0.1 mg to 2.5 mg of the aluminum-based adjuvant. In other embodiments, asingle 0.5 ml vaccine dose is formulated to contain between 0.1 mg to 2mg, 0.1 mg to 1 mg, 0.1 mg to 0.5 mg, 0.1 mg to 0.2 mg, 0.125 mg to 2.5mg, 0.125 mg to 0.5 mg, 0.125 mg to 0.2 mg or 0.125 to 0.25 mg of thealuminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccinedose is formulated to contain about 0.125 mg to about 0.250 mg of thealuminum-based adjuvant. In certain embodiments, a single 0.5 ml vaccinedose is formulated to contain about 0.125 mg of the aluminum-basedadjuvant. In certain embodiments, a single 0.5 ml vaccine dose isformulated to contain about 0.250 mg of the aluminum-based adjuvant.

In particular embodiments, the adjuvant is selected from the groupconsisting of aluminum phosphate, aluminum sulfate, and aluminumhydroxide.

In particular embodiments, the adjuvant is aluminum phosphate.

In some embodiments, the composition is for use as a vaccine against aninfection of Streptococcus pneumoniae.

Characterization of Pneumococcal Capsular Polysaccharide-Protein CarrierConjugates

In certain embodiments, the polysaccharide-protein carrier conjugate mayhave a molecular weight of 100-10,000 kDa. In certain embodiments, theconjugate has a molecular weight of 200-9,000 kDa. In certainembodiments, the conjugate has a molecular weight of 300-8,000 kDa. Incertain embodiments, the conjugate has a molecular weight of 400-7,000kDa. In certain embodiments, the conjugate has a molecular weight of500-6,000 kDa. In certain embodiments, the conjugate has a molecularweight of 600-5,000 kDa. In certain embodiments, the conjugate has amolecular weight of 500-4,000 kDa molecular weight. Any whole numberwithin any of the above ranges is contemplated as an embodiment of thepresent disclosure.

When the molecular weight is within the above range, the conjugate maybe formed stably with high yield. Also, the proportion of a freepolysaccharide can be reduced. In addition, superior immunogenicity canbe achieved within the above molecular weight range.

After the individual polysaccharide-protein conjugates are purified,they are compounded to formulate the immunogenic composition of thepresent disclosure.

The saccharide-protein conjugates of the serotypes of the presentdisclosure may be characterized by a ratio of the polysaccharide to theprotein carrier (amount of polysaccharide/amount of protein carrier,w/w).

In certain embodiments, the ratio (w/w) of the polysaccharide to theprotein carrier in the polysaccharide-protein carrier conjugate for eachserotype is 0.5-2.5, 0.4-2.3, 0.3-2.1, 0.24-2, 0.2-1.8, 0.18-1.6,0.16-1.4, 0.14-1.2, 0.12-1 or 0.1-1 (e.g., about 0.7, about 0.8, about0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5,about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about2.2, about 2.3, about 2.4 or about 2.5).

When the ratio of the polysaccharide to the protein carrier is withinthe above range, the conjugate may be formed stably with high yield.Also, the proportion of a free polysaccharide can be reduced. Inaddition, superior immunogenicity can be achieved and the conjugate canbe maintained stably without interference by other serotypes within theabove range.

The conjugates and immunogenic compositions of the present disclosuremay contain a free polysaccharide which is not covalently conjugated tothe protein carrier but is nevertheless present in thepolysaccharide-protein carrier conjugate composition. The freepolysaccharide may be non-covalently associated with thepolysaccharide-protein carrier conjugate (i.e., non-covalently bound to,adsorbed to, or entrapped in or by the polysaccharide-protein carrierconjugate).

In certain embodiments, the polysaccharide-protein carrier conjugatecontains less than about 60%, about 50%, 45%, 40%, 35%, 30%, 25%, 20% or15% of a free polysaccharide of each serotype based on the total amountof the polysaccharide of each serotype. In certain embodiments, thepolysaccharide-protein carrier conjugate of each serotype contains lessthan about 60% of a free polysaccharide of each serotype based on thetotal amount of the polysaccharide of each serotype. In certainembodiments, the polysaccharide-protein carrier conjugate of eachserotype contains less than about 50% of a free polysaccharide of eachserotype based on the total amount of the polysaccharide of eachserotype In certain embodiments, the polysaccharide-protein carrierconjugate of each serotype contains less than about 40% of a freepolysaccharide of each serotype based on the total amount of thepolysaccharide of each serotype. In certain embodiments, thepolysaccharide-protein carrier conjugate of each serotype contains lessthan about 30% of a free polysaccharide of each serotype based on thetotal amount of the polysaccharide of each serotype. In certainembodiments, the polysaccharide-protein carrier conjugate of eachserotype contains less than about 25% of a free polysaccharide of eachserotype based on the total amount of the polysaccharide of eachserotype In certain embodiments, the polysaccharide-protein carrierconjugate of each serotype contains less than about 20% of a freepolysaccharide of each serotype based on the total amount of thepolysaccharide of each serotype. In certain embodiments, thepolysaccharide-protein carrier conjugate of each serotype contains lessthan about 15% of a free polysaccharide of each serotype based on thetotal amount of the polysaccharide of each serotype In certainembodiments, the polysaccharide-protein carrier conjugate of eachserotype contains less than about 10% of a free polysaccharide of eachserotype based on the total amount of the polysaccharide of eachserotype.

The polysaccharide-protein carrier conjugate of each serotype may alsobe characterized by its molecular size distribution (K_(d)). A sizeexclusion chromatography medium (CL-4B; cross-linked agarose beads, 4%)may be used to determine the relative molecular size distribution of theconjugate. Size exclusion chromatography (SEC) is used in a gravity-fedcolumn to profile the molecular size distribution of the conjugate.Large molecules excluded from the pores in the medium are eluted morequickly than small molecules. A fraction collector is used to collectthe column eluate. The fractions are tested colorimetrically bysaccharide assay. For the determination of K_(d), the column iscalibrated to establish the fraction at which molecules are completelyexcluded (V₀; K_(d)=0) and the fraction representing the maximumretention (V_(i); K_(d)=1). The fraction at which a specified sampleattribute is reached (V_(e)) is related to K_(d) by the expressionK_(d)=(V_(e)−V₀)/(V_(i)−V₀).

In certain embodiments, at least 15% of the polysaccharide-proteincarrier conjugate of each serotype may have a K_(d) of 0.3 or below in aCL-4B column.

In certain embodiments, at least 20% of the polysaccharide-proteincarrier conjugate of each serotype may have a K_(d) of 0.3 or below in aCL-4B column. In certain embodiments, at least 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of thepolysaccharide-protein carrier conjugate of each serotype may have aK_(d) of 0.3 or below in a CL-4B column. In certain embodiments, atleast 60% of the polysaccharide-protein carrier conjugate of eachserotype may have a K_(d) of 0.3 or below in a CL-4B column. In certainembodiments, at least 50-80% of the polysaccharide-protein carrierconjugate of each serotype may have a K_(d) of 0.3 or below in a CL-4Bcolumn. In certain embodiments, at least 65-80% of thepolysaccharide-protein carrier conjugate of each serotype may have aK_(d) of 0.3 or below in a CL-4B column. In certain embodiments, atleast 15-60% of the saccharide-protein conjugate of each serotype mayhave a K_(d) of 0.3 or below in a CL-4B column.

Prophylactic Methods and Uses

In one aspect, this disclosure provides a vaccine comprising a mixedcarrier, 21-valent pneumococcal conjugate composition and apharmaceutically acceptable excipient. In some embodiments, thepharmaceutically acceptable excipient comprises at least a buffer, suchas a succinate buffer, a salt, such as sodium chloride, and/or a surfaceactive agent, such as a polyoxyethylene sorbitan ester (e.g.,polysorbate 80). In some embodiments, two polysaccharides selected fromthe group consisting of serotypes 1, 3, and 5 are conjugated to thetetanus toxoid, and the remaining capsular polysaccharides among 1, 3,4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F,23F, and 33F are conjugated to CRM₁₉₇ (21-valent).

In one embodiment, the capsular polysaccharides from serotypes 1 and 5are conjugated to the tetanus toxoid, and the capsular polysaccharidesfrom serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇ (21-valent).

In another embodiment, the capsular polysaccharides from serotypes 1 and3 are conjugated to the tetanus toxoid, and the capsular polysaccharidesfrom serotypes 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇ (21-valent).

In another embodiment, the capsular polysaccharides from serotypes 3 and5 are conjugated to the tetanus toxoid, and the capsular polysaccharidesfrom serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇ (21-valent).

In some embodiments, the vaccine elicits a protective immune response ina human subject against disease caused by Streptococcus pneumoniaeinfection.

According to a further aspect, this disclosure provides a method forprophylaxis of Streptococcus pneumoniae infection or disease, the methodcomprising administering to a human subject a prophylactically effectiveamount of a mixed carrier, 21-valent pneumococcal conjugate compositionor a vaccine comprising the same. The mixed carrier, 21-valentpneumococcal conjugate composition or vaccine comprising the same may beadministered by any route, including, for example, by a systemic ormucosal route, as described below in further detail.

In certain embodiments, the human subject is an elderly subject and thedisease is pneumonia or invasive pneumococcal disease (IPD). In certainembodiments, the elderly subject is at least 50 years old. In otherembodiments, the elderly subject is at least 55 years old. In yet otherembodiments, the elderly subject is at least 60 years old.

In other embodiments, the human subject is an infant and the disease ispneumonia, invasive pneumococcal disease (IPD), or acute otitis media(AOM). In certain embodiments, the infant is 0-2 years. In otherembodiments, the infant is 2 to 15 months.

In yet another embodiment, the human subject is 6 weeks to 17 years ofage and the disease is pneumonia, invasive pneumococcal disease (IPD) oracute otitis media (AOM). In certain embodiments, the human subject is 6weeks to 5 years of age. In other embodiments, the human subject is 5 to17 years of age.

The amount of conjugate in each vaccine dose or the prophylacticallyeffective amount of the mixed carrier, multivalent pneumococcalconjugate composition may be selected as an amount that inducesprophylaxis without significant, adverse effects. Such an amount canvary depending upon the pneumococcal serotype. Generally, each dose mayinclude about 0.1 μg to about 100 μg of polysaccharide, specifically,about 0.1 to 10 μg, and, more specifically, about 1 μg to about 5 μg.Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, the amount for vaccination ofa human subject can be determined by extrapolating an animal testresult. In addition, the dose can be determined empirically.

In some embodiments, the vaccine or the mixed carrier, 21-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 1 μg to about 5 μg of each capsularpolysaccharide; about 1 μg to about 30 μg of TT; about 20 μg to about 85μg of CRM₁₉₇; and optionally about 0.1 mg to about 0.5 mg of elementalaluminum adjuvant. In some embodiments, the vaccine or the mixedcarrier, 21-valent pneumococcal conjugate composition may be a single0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of eachcapsular polysaccharide except serotype 6B and optionally serotype 3,which is/are present in an amount of about 4 μg to about 5 μg; about 2μg to about 25 μg of TT; about 40 μg to about 75 μg of CRM₁₉₇; andoptionally about 0.1 mg to about 0.25 mg of elemental aluminum adjuvant.

In some embodiments, the vaccine or the mixed carrier, 21-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 2.2 μg of each capsular polysaccharideexcept serotype 6B, which is present in an amount of about 4.4 μg.

In some embodiments, the vaccine or the mixed carrier, 21-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 2 μg to about 2.5 μg of each capsularpolysaccharide except for up to six capsular polysaccharides selectedfrom the group consisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F,each of which is present in an amount of about 4 μg to about 5 μg. Inone embodiment, the up to six capsular polysaccharides, present in anamount of about 4 μg to about 5 μg, are selected from the groupconsisting of serotypes 1, 3, 4, 6B, 9V, 19A, and 19F. In otherembodiments, the vaccine or the mixed carrier, 21-valent pneumococcalconjugate composition may be a single 0.5 ml dose formulated to containabout 2.2 μg of each capsular polysaccharide except for up to sixcapsular polysaccharides selected from the group consisting of serotypes1, 3, 4, 5, 6B, 9V, 19A, and 19F, each of which is present in an amountof about 4.4 μg. In one embodiment, the up to six capsularpolysaccharides, present in an amount of about 4.4 μg, are selected fromthe group consisting of serotypes 1, 3, 4, 6B, 9V, 19A, and 19F.

In some embodiments, the vaccine or the mixed carrier, 21-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 2 μg to about 2.5 μg of the capsularpolysaccharides of serotypes 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14,15B, 18C, 22F, 23F, and 33F and about 4 μg to about 5 μg of the capsularpolysaccharides of serotypes 3, 4, 6B, 9V, 19A, and 19F.

In certain embodiments, the vaccine or the mixed carrier, 21-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 2 to about 2.5 μg of the capsularpolysaccharides of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F and about 4 to about 5 μg ofthe capsular polysaccharides of serotypes 3 and 6B.

In some embodiments, the vaccine or the mixed carrier, 21-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 2 to about 2.5 μg of the capsularpolysaccharides of serotypes 1, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F and about 4 to about 5 μg ofthe capsular polysaccharides of serotype 6B and about 8 to about 9 μg ofthe capsular polysaccharides of serotype 3, and more preferably about8.8 μg of the capsular polysaccharides of serotype 3.

In certain embodiments, the mixed carrier, 21-valent pneumococcalconjugate composition or vaccine comprising the same further comprisessodium chloride and sodium succinate buffer as excipients.

In some embodiments, the mixed carrier, 21-valent pneumococcal conjugatecomposition may be formulated into a liquid formulation in which each ofthe pneumococcal capsular polysaccharides of serotypes 1 and 3 isconjugated to TT and the capsular polysaccharides from serotypes 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F,and 33F are conjugated to CRM₁₉₇ (21-valent). Each 0.5 mL dose may beformulated into a liquid containing: about 2.2 μg of each capsularpolysaccharide, except for serotype 6B at about 4.4 μg; about 2 μg toabout 25 μg of TT carrier protein (only for the serotypes 1 and 3) andabout 40 μg to about 75 μg of CRM₁₉₇ carrier protein; about 0.125 to0.250 mg of elemental aluminum (about 0.5 to about 1.2 mg aluminumphosphate) as an adjuvant; and sodium chloride and sodium succinatebuffer as excipients.

In some embodiments, the mixed carrier, 21-valent pneumococcal conjugatecomposition may be formulated into a liquid formulation in which each ofthe pneumococcal capsular polysaccharides of serotypes 1 and 5 isconjugated to TT and the capsular polysaccharides from serotypes 3, 4,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F,and 33F are conjugated to CRM₁₉₇ (21-valent). In one embodiment, each0.5 mL dose may be formulated into a liquid containing: about 2.2 μg ofeach capsular polysaccharide, except for serotype 6B at about 4.4 μg andserotype 3 at about 2.2-8.8 μg; about 2 μg to about 25 μg of TT carrierprotein (only for the serotypes 1 and 5) and about 40 μg to about 75 μgof CRM₁₉₇ carrier protein; about 0.125 to 0.250 mg of elemental aluminum(about 0.5 to 1.2 mg aluminum phosphate) adjuvant; and sodium chlorideand sodium succinate buffer as excipients. In certain embodiments,serotype 3 is present at about 2.2 μg. In other embodiments, serotype 3is present at about 4.4 μg. In other embodiments, serotype 3 is presentat about 8.8 μg. In yet another embodiment, each 0.5 mL dose may beformulated into a liquid containing: about 2.2 μg of each capsularpolysaccharide, except for up to six capsular polysaccharides selectedfrom the group consisting of serotype 1, 3, 4, 5, 6B, 9V, 19A, and 19Fat about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (onlyfor the serotypes 1 and 5) and about 40 μg to about 75 μg of CRM₁₉₇carrier protein; about 0.125 mg to 0.250 mg of elemental aluminum (0.5mg to 1.2 mg aluminum phosphate) adjuvant; and sodium chloride andsodium succinate buffer as excipients. In one embodiment, the up to sixcapsular polysaccharides at about 4.4 μg are selected from the groupconsisting of serotype 1, 3, 4, 6B, 9V, 19A, and 19F. In anotherembodiment, each 0.5 mL dose may be formulated into a liquid containing:about 2.2 μg of each capsular polysaccharide, except for serotypes 3, 4,6B, 9V, 19A, and 19F at about 4.4 μg; about 2 μg to about 25 μg of TTcarrier protein (only for the serotypes 1 and 5) and about 40 μg toabout 75 μg of CRM₁₉₇ carrier protein; about 0.125 mg to 0.250 mg ofelemental aluminum (0.5 mg to 1.2 mg aluminum phosphate) adjuvant; andsodium chloride and sodium succinate buffer as excipients. In anotherembodiment, each 0.5 mL dose may be formulated into a liquid containing:about 2.2 μg of each capsular polysaccharide, except for serotypes 3 and4 at about 4.4 μg; about 2 μg to about 25 μg of TT carrier protein (onlyfor the serotypes 1 and 5) and about 40 μg to about 75 μg of CRM₁₉₇carrier protein; about 0.125 mg to 0.250 mg of elemental aluminum (0.5mg to 1.2 mg aluminum phosphate) adjuvant; and sodium chloride andsodium succinate buffer as excipients.

In some embodiments, the mixed carrier, 21-valent pneumococcal conjugatecomposition may be formulated into a liquid formulation in which each ofthe pneumococcal capsular polysaccharides of serotypes 3 and 5 isconjugated to TT and the capsular polysaccharides from serotypes 1, 4,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F,and 33F are conjugated to CRM₁₉₇ (21-valent). Each 0.5 mL dose may beformulated into a liquid containing: about 2.2 μg of each capsularpolysaccharide, except for 6B at about 4.4 μg; about 2 μg to about 25 μgof TT carrier protein (only for the serotypes 3 and 5) and about 40 μgto about 75 μg of CRM₁₉₇ carrier protein; about 0.125 to 0.250 mg ofelemental aluminum (about 0.5 to 1.2 mg aluminum phosphate) adjuvant;and sodium chloride and sodium succinate buffer as excipients.

In some embodiments, the liquid formulation may be filled into a singledose syringe without a preservative. After shaking, the liquidformulation becomes a vaccine that is a homogeneous, white suspensionready for intramuscular administration.

The mixed carrier, 21-valent pneumococcal conjugate composition can beadministered in a single injection or as part of an immunization series.For example, the mixed carrier, 21-valent pneumococcal conjugatecomposition can be administered 2, 3, 4, or more times at appropriatelyspaced intervals, such as, a 1, 2, 3, 4, 5, or 6 month interval or acombination thereof. In some embodiments, the mixed carrier, 21-valentpneumococcal conjugate composition is administered to an infant 4 timeswithin the first 15 months of birth, including, for example, at about 2,3, 4, and 12-15 months of age; at about 3, 4, 5, and 12-15 months ofage; or at about 2, 4, 6, and 12-15 months of age. This first dose canbe administered as early as 6 weeks of age. In another embodiment, themixed carrier, 21-valent pneumococcal conjugate composition isadministered to an infant 3 times within the first 15 months of birth,including, for example, at about 2, 4, and 11-12 months.

The mixed carrier, multivalent pneumococcal conjugate composition mayalso include one or more proteins from Streptococcus pneumoniae.Examples of Streptococcus pneumoniae proteins suitable for inclusioninclude those identified in International Patent ApplicationWO02/083855, as well as those described in International PatentApplication WO02/053761.

The mixed carrier, 21-valent pneumococcal conjugate composition can beadministered to a subject via one or more administration routes known toone of ordinary skill in the art such as a parenteral, transdermal, ortransmucosal, intranasal, intramuscular, intraperitoneal,intracutaneous, intravenous, or subcutaneous route and be formulatedaccordingly. The mixed carrier, 21-valent pneumococcal conjugatecomposition can be formulated to be compatible with its intended routeof administration.

In some embodiments, the mixed carrier, 21-valent pneumococcal conjugatecomposition can be administered as a liquid formulation byintramuscular, intraperitoneal, subcutaneous, intravenous,intraarterial, or transdermal injection or respiratory mucosalinjection. The mixed carrier, 21-valent pneumococcal conjugatecompositions can be formulated in liquid form or in a lyophilized form.In some embodiments, injectable compositions are prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. In some embodiments, injection solutions andsuspensions are prepared from sterile powders or granules. Generalconsiderations in the formulation and manufacture of pharmaceuticalagents for administration by these routes may be found, for example, inRemington's Pharmaceutical Sciences, 19^(th) ed., Mack Publishing Co.,Easton, Pa., 1995; incorporated herein by reference. At present the oralor nasal spray or aerosol route (e.g., by inhalation) are most commonlyused to deliver therapeutic agents directly to the lungs and respiratorysystem. In some embodiments, a mixed carrier, 21-valent pneumococcalconjugate composition is administered using a device that delivers ametered dosage of composition. Suitable devices for use in deliveringintradermal pharmaceutical compositions described herein include shortneedle devices such as those described in U.S. Pat. Nos. 4,886,499,5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496,5,417,662 (all of which are incorporated herein by reference).Intradermal compositions may also be administered by devices which limitthe effective penetration length of a needle into the skin, such asthose described in WO1999/34850, incorporated herein by reference, andfunctional equivalents thereof. Also suitable are jet injection deviceswhich deliver liquid vaccines to the dermis via a liquid jet injector orvia a needle which pierces the stratum corneum and produces a jet whichreaches the dermis. Jet injection devices are described for example inU.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912,5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163,5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824,4,941,880, 4,940,460, WO1997/37705, and WO1997/13537 (all of which areincorporated herein by reference). Also suitable are ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis. Additionally, conventional syringes may be used in the classicalMantoux method of intradermal administration.

Preparations for parenteral administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol, oilssuch as olive oil, and injectable organic esters such as ethyl oleate.Examples of oil include vegetable or animal oil, peanut oil, soybeanoil, olive oil, sunflower oil, liver oil, synthetic oil such as marineoil, and lipids obtained from milk or eggs. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

The mixed carrier, 21-valent pneumococcal conjugate composition can beformulated in the form of a unit dose vial, multiple dose vial, orpre-filled syringe. A pharmaceutically acceptable carrier for a liquidformulation includes aqueous or nonaqueous solvent, suspension,emulsion, or oil. The composition may be isotonic, hypertonic, orhypotonic. However, it is desirable that the composition for infusion orinjection is basically isotonic. Thus, isotonicity or hypertonicity maybe advantageous for storage of the composition. When the composition ishypertonic, the composition can be diluted to isotonicity beforeadministration. A tonicity agent may be ionic tonicity agent such assalt or non-ionic tonicity agent such as carbohydrate. The ionictonicity agent includes, but is not limited to, sodium chloride, calciumchloride, potassium chloride, and magnesium chloride. The nonionictonicity agent includes, but is not limited to, sorbitol and glycerol.Preferably, at least one pharmaceutically acceptable buffer is included.For example, when the composition is an infusion or injection, it ispreferable to be formulated in a buffer with a buffering capacity at pH4 to pH 10, such as pH 5 to pH 9, or, pH 6 to pH 8. The buffer may beselected from those suitable for United States Pharmacopeia (USP). Forexample, the buffer can be selected from the group consisting of amonobasic acid, such as acetic acid, benzoic acid, gluconic acid,glyceric acid, and lactic acid; a dibasic acid, such as aconitic acid,adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid,succinic acid, and tartaric acid; a polybasic acid such as citric acidand phosphoric acid; and a base such as ammonia, diethanolamine,glycine, triethanolamine, and TRIS.

The mixed carrier, 21-valent pneumococcal conjugate composition maycomprise a surface active agent. Examples of the surface active agentinclude, but are not limited to, polyoxyethylene sorbitan ester(generally referred to as Tweens), in particular, polysorbate 20 andpolysorbate 80; copolymers (such as DOWFAX) of ethylene oxide (EO),propylene oxide (PO), butylenes oxide (BO); octoxynols with differentrepeats of ethoxy(oxy-1,2-ethanediyl) group, in particular, octoxynol-9(Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630/NP-40);phospholipid such as lecithin; nonylphenol ethoxylate such as TERGITOLNP series; lauryl, cetyl, stearyl, oleyl alcohol-derived polyoxyethylenefatty ether (Brij surfactant), in particular, triethyleneglycolmonolauryl ether (Brij 30); sorbitan ether known as SPAN, in particular,sorbitan trioleate (Span 85) and sorbitan monolaurate.

Mixtures of surface active agents such as Tween 80/Span 85 can be used.A combination of polyoxyethylene sorbitan ester such as Tween 80 andoctoxynol such as Triton X-100 is also suitable. A combination ofLaureth 9 and Tween and/or octoxynol is also advantageous. Preferably,the amount of polyoxyethylene sorbitan ester (such as Tween 80) includedmay be 0.01% to 1% (w/v), 0.01% to 0.1% (w/v), 0.01% to 0.05% (w/v), orabout 0.02%; the amount of octylphenoxy polyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton X-100) included may be 0.001% to 0.1%(w/v), in particular 0.005% to 0.02%; and the amount of polyoxyethyleneether (such as Laureth 9) included may be 0.1% to 20% (w/v), possibly0.1% to 10%, in particular 0.1% to 1% or about 0.5%.

In some embodiments, the mixed carrier, 21-valent pneumococcal conjugatecomposition may be delivered via a release control system. For example,intravenous infusion, transdermal patch, liposome, or other routes canbe used for administration. In one aspect, macromolecules such asmicrosphere or implant can be used.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theinvention.

Examples Example 1. Preparation of S. pneumoniae CapsularPolysaccharides

Cultivation of S. pneumoniae and purification of capsularpolysaccharides were conducted as known to one of skill in the art. S.pneumoniae serotypes were obtained from the American Type CultureCollection (ATCC) (serotype 1: ATCC No. 6301; serotype 3: ATCC No. 6303;serotype 4: ATCC No. 6304; serotype 5: ATCC No. 6305; serotype 6A: ATCCNo. 6306; serotype 6B: ATCC No. 6326; serotype 7F: ATCC No. 10351;serotype 9N: ATCC No. 6309; serotype 9V: ATCC No. 10368; serotype 14:ATCC No. 6314; serotype 18C: ATCC No. 10356; serotype 19A: ATCC No.10357; serotype 19F: ATCC No. 6319; serotype 23F: ATCC No. 6323).Internal strains for serotypes 8, 10A, 11A, 12F, 15B, 22F, and 33F wereused, but any publically available strain can be used. S. pneumoniaewere characterized by capsules and motility, Gram-positive,lancet-shaped diplococcus, and alpha hemolysis in a blood agar medium.Serotypes were identified by Quelling test using specific anti-sera(U.S. Pat. No. 5,847,112).

Preparation of Cell Banks

Several generations of seed stocks were generated in order to expand thestrains and remove components of animal origin (generations F1, F2, andF3). Two additional generations of seed stocks were produced. The firstadditional generation was cultured from an F3 vial, and the subsequentgeneration was cultured from a vial of the first additional generation.Seed vials were stored frozen (below −70° C.) with synthetic glycerol asa cryopreservative. For cell bank preparation, all cultures were grownin a soy-based medium. Prior to freezing, cells were concentrated bycentrifugation, spent medium was removed, and cell pellets werere-suspended in a fresh medium containing a cryopreservative (such assynthetic glycerol).

Culturing and Harvesting

Cultures from the working cell bank were inoculated into seed bottlescontaining a soy-based medium and cultured. After the target opticaldensity (absorbance) was reached, the seed bottle was used to inoculatea fermentor containing the soy-based medium. The culturing wasterminated when an optical density value started to be maintainedconstant. After terminating the culturing, sodium deoxycholate was addedto the culture to lyse the cells. The resulting fermentor contents werecooled, and protein precipitation was induced. Then, the mixture wascentrifuged to remove precipitated proteins and cell debris.

Purification

The solution obtained from the centrifugation was filtered through adepth filter to remove the proteins and cell debris that had notprecipitated in the centrifugation. The filtrate was concentrated on a100 kDa MW membrane and the concentrate was diafiltered with 10 volumesof a 25 mM sodium phosphate buffer (pH 7.2) to obtain a sample. Thesample was filtered to collect a supernatant from which polysaccharideswere precipitated and filtered. The filtrate was concentrated on a 30kDa membrane, and the concentrate was diafiltered using about 10 volumesof triple distilled water. After performing the diafiltration, theremaining solution was filtered through a 0.2 μm filter. An in-processcontrol test was performed on the filtrate (appearance, remainingproteins, remaining nucleic acids, endotoxins, molecular weights, andthe total amount of polysaccharides). The concentrate was sterilefiltered and stored at −20° C.

Example 2. Preparation of Conjugate of S. pneumoniae CapsularPolysaccharide and Carrier Protein

Polysaccharides of different serotypes were activated followingdifferent pathways and then conjugated to a carrier protein, CRM₁₉₇ orTT. Specifically, conjugates were prepared by conjugating each of thecapsular polysaccharides of all serotypes to CRM₁₉₇ and by conjugatingeach of the capsular polysaccharides of the serotypes 1, 3, and 5 to TT.Depending on the size of the native serotype the activation process mayinclude reduction of the size of each capsular polysaccharide to thetarget molecular weight, chemical activation, and buffer exchange viaultrafiltration. The conjugates were purified using ultrafiltration andfinally filtered through 0.2 μm filter. The process parameters such aspH, temperature, concentration, and time were as follows.

(1) Activation Process

Step 1: Hydrolysis

Reductive amination is a known method for conjugating polymers in whichan amide bond is formed between a primary amine (—NH₂) group of aprotein and an aldehyde of a saccharide. Aldehyde groups are added tothe pneumococcal capsular polysaccharide to promote conjugation to thecarrier protein. A vicinal diol structure of a monosaccharide can beoxidized by sodium periodate (NaIO₄) to form aldehyde groups. Thecapsular polysaccharides from serotypes 1, 3, 4, 6A, 8, 11A, 12F, 14,15B, 18C, 22F, and 33F were pre-treated as follows.

In the case of the serotype 1, sodium hydroxide (at a final baseconcentration of 0.05 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 50±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and hydrochloric acid was added thereto to a final pH of 6.0±0.1,thereby stopping hydrolysis.

In the case of the serotype 3, 8, 11A, and 15B, hydrochloric acid (at afinal acid concentration of 0.01 M) was added to a solution of thecapsular polysaccharide, and the solution was incubated at 60±2° C. Thesolution was then cooled to a temperature in a range of about 21° C. toabout 25° C., and 0.1M sodium phosphate was added thereto to a final pHof 6.0±0.1, thereby stopping hydrolysis.

In the case of the serotype 4, hydrochloric acid (at a final acidconcentration of 0.1 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 45±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 1M sodium phosphate was added thereto to a final pH of 6.0±0.1,thereby stopping hydrolysis.

In the case of the serotype 6A, glacial acetic acid (at a final acidconcentration of 0.1 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 60±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 1M sodium hydroxide was added thereto to a final pH of 6.0±0.1,thereby stopping hydrolysis.

In the case of the serotype 12F, hydrochloric acid (at a final acidconcentration of 0.01 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 70±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 0.1M sodium phosphate was added thereto to a final pH of thesolution of 6.0±0.1, thereby stopping hydrolysis.

In the case of the serotypes 14 and 18C, glacial acetic acid (at a finalacid concentration of 0.2 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 94±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 1M sodium phosphate was added thereto so that a final pH of thesolution was 6.0±0.1, thereby stopping hydrolysis.

In the case of the serotypes 22F and 33F, hydrochloric acid (at a finalacid concentration of 0.01 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 60±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 0.1M sodium phosphate was added thereto to a final pH of6.0±0.1, thereby stopping hydrolysis.

Each of the obtained capsular polysaccharides was diluted in water forinjection (WFI), sodium acetate, and sodium phosphate to a finalconcentration between about 1.0 mg/mL and about 2.0 mg/mL.

Step 2: Periodate Reaction

The sodium periodate molar equivalent for each pneumococcal saccharideactivation was determined based on repeating unit molar mass. Withthorough mixing, the oxidation reaction was allowed to proceed for 16 to20 hours at 21° C. to 25° C. for all serotypes except for 1, 7F, and19F, for which the temperature was 10° C. or less. To help maintainconsistent and stable production of conjugates, a range of degree ofoxidation (Do) levels for each serotype is targeted during theconjugation process. A preferred, targeted range for the Do levels foreach serotype is shown in Table 1 and Table 2.

TABLE 1 Range of Do for all serotypes to be conjugated to CRM₁₉₇Serotype Range of Do Serotype Range of Do Serotype 1 4 to 10 Serotype11A 1 to 15 Serotype 3 2 to 8 Serotype 12F 1 to 9 Serotype 4 1 to 5Serotype 14 6 to 13 Serotype 5 2 to 6 Serotype 15B 1 to 17 Serotype 6A 5to 15 Serotype 18C 6 to 14 Serotype 6B 7 to 13 Serotype 19A 7 to 13Serotype 7F 2 to 8 Serotype 19F 6 to 12 Serotype 8 1 to 17 Serotype 22F1 to 16 Serotype 9N 5 to 10 Serotype 23F 6 to 14 Serotype 9V 4 to 9Serotype 33F 1 to 15 Serotype 10A 1 to 12

TABLE 2 Range of Do for serotypes 1, 3, and 5 to be conjugated to TTSerotype Range of Do Serotype 1 (1-TT) 1 to 15 Serotype 3 (3-TT) 2 to 14Serotype 5 (5-TT) 1 to 15

Step 3: Ultrafiltration

The oxidized saccharide was concentrated and diafiltered with WFI on a100 kDa MWCO ultrafilter (30 kDa ultrafilter for serotype 1 and 5 kDaultrafilter for serotype 18C). Diafiltration was conducted using 0.9%sodium chloride solution for serotype 1, 0.01 M sodium acetate buffer(pH 4.5) for serotype 7F and 23F, and 0.01 M sodium phosphate buffer (pH6.0) for serotype 19F. The permeate was discarded, and the retentate wasfiltered through a 0.2 μm filter.

Step 4: Lyophilization

For capsular polysaccharides of serotypes 3, 4, 5, 8, 9N, 9V, 10A, 14,15B, 22F, and 33F that are to be conjugated to a carrier protein byusing an aqueous solvent, a mixed solution of polysaccharides andcarrier protein was prepared without adding further sucrose,lyophilized, and then stored at −25° C.±5° C.

For capsular polysaccharides of serotypes 1 and 18C that are to beconjugated to a carrier protein by using an aqueous solvent,polysaccharides and carrier protein were independently prepared, withoutadding further sucrose, lyophilized, and then stored at −25° C.±5° C.

For capsular polysaccharides of serotypes 6A, 6B, 7F, 19A, 19F, and 23Fthat are to be conjugated to a carrier protein by using a DMSO solvent,a predetermined amount of sucrose to reach a final sucrose concentrationof 5%±3% (w/v) was added to the activated saccharides, and the sampleswere independently prepared, lyophilized, and then stored at −25° C.±5°C.

For capsular polysaccharide of serotype 11A, a predetermined amount ofsucrose to reach a final sucrose concentration of 20%±5% (w/v) was addedto the activated saccharide, and the polysaccharides and carrier proteinwere independently prepared, lyophilized, and then stored at −25° C.±5°C.

For capsular polysaccharide of serotype 12F, a predetermined amount ofsucrose to reach a final sucrose concentration of 10%±5% (w/v) was addedto the activated saccharide, and the polysaccharides and carrier proteinwere independently prepared, lyophilized, and then stored at −25° C.±5°C.

(2) Conjugation Process

Aqueous conjugation was conducted for serotypes 1, 3, 4, 5, 8, 9N, 9V,10A, 14, 15B, 18C, 22F, and 33F, and DMSO conjugation was conducted forserotypes 6A, 6B, 7F, 11A, 12F, 19A, 19F, and 23F. Each of the capsularpolysaccharides was conjugated to a carrier protein at a ratio of 0.2 to2:1.

Step 1: Dissolution

Aqueous Conjugation

For serotypes 1, 3, 4, 5, 8, 9N, 9V, 10A, 14, 15B, 18C, 22F, and 33F,the lyophilized sample was thawed and equilibrated at room temperature.The lyophilized sample was reconstituted to a reaction concentration byusing a sodium phosphate buffer solution at 23±2° C. at a ratio set foreach serotype.

Dimethyl Sulfoxide (DMSO) Conjugation

For serotypes 6A, 6B, 7F, 11A, 12F, 19A, 19F, and 23F, the lyophilizedsample was thawed, equilibrated at room temperature, and reconstitutedin DMSO.

Step 2: Conjugation Reaction

Aqueous Conjugation

For serotypes 3-TT, 4, 5, 5-TT, 8, 9N, 9V, 10A, 14, 15B, 18C, 22F, and33F, the conjugation reaction was initiated by adding the sodiumcyanoborohydride solution (100 mg/mL) to 1.0 to 1.4 moles sodiumcyanoborohydride per mole of saccharide. However, for serotypes 1, 1-TTand 3, the reaction was initiated by adding the sodium cyanoborohydridesolution to 0.5 moles sodium cyanoborohydride per mole of saccharide.

The reaction mixture was incubated at 23° C. to 37° C. for 44 to 106hours. The reaction temperature and time were adjusted by serotype. Thetemperature was then reduced to 23±2° C. and sodium chloride 0.9% wasadded to the reactor. Sodium borohydride solution (100 mg/mL) was addedto achieve 1.8 to 2.2 molar equivalents of sodium borohydride per moleof saccharide. The mixture was incubated at 23±2° C. for 3 to 6 hours.This procedure reduced any unreacted aldehydes present on thesaccharides. Then, the mixture was diluted with sodium chloride 0.9% andthe diluted conjugation mixture was filtered using a 0.8 or 0.45 μmpre-filter.

DMSO Conjugation

For capsular polysaccharides of serotypes 6A, 6B, 7F, 11A, 12F, 19A,19F, and 23F, the conjugation reaction was initiated by adding thesodium cyanoborohydride solution (100 mg/mL) to a ratio of 0.8 to 1.2molar equivalents of sodium cyanoborohydride per one mole of activatedsaccharide. WFI was added to the reaction mixture to a targetconcentration of 1% (v/v), and the mixture was incubated for 12 to 26hours at 23±2° C. 100 mg/mL of a sodium borohydride solution (typical1.8 to 2.2 molar equivalents sodium borohydride per mole activatedsaccharide) and WFI (target 5% v/v) were added to the reaction and themixture was incubated for 3 to 6 hours at 23±2° C. This procedurereduced any unreacted aldehydes present on the saccharides. Then, thereaction mixture was diluted with sodium chloride 0.9%, and the dilutedconjugation mixture was filtered using a 0.8 or 0.45 μm pre-filter.

Step 3: Ultrafiltration

The diluted conjugate mixture was concentrated and diafiltered on a 100kDa MWCO ultrafiltration filter or a 300 kDa MWCO ultrafiltration filterwith a minimum of 15 volumes of 0.9% sodium chloride or buffer. Also,the composition and pH of the buffer used in the process varieddepending on each of the serotypes.

Step 4: Sterile Filtration

The retentate after the ultrafiltration was sterile filtered (0.2 μm),and in-process controls (appearance, free protein, free saccharide,molecular size distribution, sterility, saccharide content, proteincontent, pH, endotoxin, residual cyanide, residual DMSO, saccharideidentity, TT identity, and CRM₁₉₇ identity) were performed on thefiltered conjugates. The final concentrate was refrigerated and storedat 2° C. to 8° C.

Example 3. Formulation of Multivalent Pneumococcal Conjugate Vaccine

The desired volumes of final bulk concentrates obtained from Example 2were calculated based on the batch volume and the bulk saccharideconcentrations. After the 0.85% sodium chloride (physiological saline),polysorbate 80, and succinate buffer were added to the pre-labeledformulation vessel, bulk concentrates were added. The preparation wasthen thoroughly mixed and sterile filtered through a 0.2 μm membrane.The formulated bulk was mixed gently during and following the additionof bulk aluminum phosphate. The pH was checked and adjusted ifnecessary. The formulated bulk product was stored at 2 to 8° C. Thefollowing, non-limiting, multivalent pneumococcal conjugate vaccineformulations were prepared and named PCV21(1/5)-TT and PCV21(3/5)-TT:

PCV21(1/5)-TT included polysaccharide-conjugates prepared by conjugatingeach polysaccharide of the serotypes 1 and 5 to TT and eachpolysaccharide of the serotypes 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F to CRM₁₉₇; and

PCV21(3/5)-TT included polysaccharide-conjugates prepared by conjugatingeach polysaccharide of the serotypes 3 and 5 to TT and eachpolysaccharide of the serotypes 1, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F to CRM₁₉₇.

The PCV21(1/5)-TT composition in a total dose of 0.5 ml included 2.2 μgof each polysaccharide, except for serotype 6B at 4.4 μg; 2 μg to 25 μgof TT (for serotypes 1 and 5) and 40 μg to 75 μg of CRM₁₉₇; 0.125 mg ofelemental aluminum (0.5 mg aluminum phosphate) adjuvant; 4.25 mg ofsodium chloride; about 295 μg of a succinate buffer solution; and about100 μg of polysorbate 80 in the total of 0.5 ml dose. In certainexperiments, the amount of serotype 3 polysaccharide was increased to4.4 μg or 8.8 μg in the PCV21(1/5)-TT composition. In certainexperiments, the amount of polysaccharide for each of serotypes 3, 4,6B, 9V, 19A, and 19F was increased to 4.4 μg in the PCV21(1/5)-TTcomposition.

The PCV21(3/5)-TT composition in a total dose of 0.5 ml included 2 μg to25 μg of TT (for serotypes 3 and 5) and 40 μg to 75 μg CRM₁₉₇,respectively, with the other components and contents thereof identicalto those of PCV21(1/5)-TT. In certain compositions, the amount ofelemental aluminum in the 0.5 ml dose was increased to 0.250 mg.

Example 4. Immunogenicity of Multivalent Pneumococcal Conjugate Vaccine

The mixed carrier, multivalent pneumococcal vaccines, PCV21(1/5)-TT andPCV21(3/5)-TT prepared in Example 3, were tested for the ability toinduce an immunogenic response in rabbits. Immunogenicity assessment wasperformed by antigen-specific ELISA for serum IgG concentrations and byopsonophagocytic assay (OPA) for antibody functionality. New ZealandWhite rabbits were immunized intramuscularly at week 0 and week 2 with adose of 5% higher than the planned human clinical dose of eachpolysaccharide (2.31 μg of each polysaccharide, except for 6B at 4.62μg) in the formulation or the human dose (2.2 ug of each polysaccharide,except for 6B at 4.4 ug). Sera were sampled every 2 weeks postimmunization. Both concentrations showed the same results.

4-1. PCV21(3/5)-TT

Serotype Specific IgG Concentration Measurement

Capsular polysaccharides (PnPs) for each serotype were coated on a96-well plate at 0.5 μg/well to 1 μg/well. An equivalent amount of serumwas sampled from each subject and was pooled by group. The serum poolwas serially diluted by 2.5 times with an antibody dilution buffercomprising Tween 20 and pneumococcal cell-wall polysaccharide (CWPS)obtained from Statens Serum Institut (5 μg/mL) and then reacted at roomtemperature for 30 minutes. The plate was washed 5 times with a washingbuffer and then pre-adsorbed and diluted serum 50 μl was added to thecoated well plate, followed by incubation at room temperature for 2hours to 18 hours. The well plate was washed in the same way and thengoat anti-Rabbit IgG-alkaline phosphatase conjugates were added to eachwell, followed by incubation at room temperature for 2 hours. Plateswere washed as described above and 1 mg/mL p-nitrophenylamine buffer assubstrate was added to each well and then reacted at room temperaturefor 2 hours. The reaction was quenched by adding 50 μl of 3 M NaOH andabsorbances at 405 nm and 690 nm were measured. As a comparativeexample, the commercially available, 13-valent vaccine (PREVNAR13) wassubjected to the same procedure. The results are shown in Table 3.

TABLE 3 IgG concentration (U/mL) for 21 serotypes at 2 weeks aftersecondary immunization PCV21(3/5)-TT PCV21(3/5)-TT Serotype PREVNAR130.125 mg Al 0.250 mg Al 1 16770.4 15320.6 38499.1 3 6603.4 30132.832029.7 4 27969.9 51034.4 71638.3 5 5758.6 15627.5 11044.7 6A 9493.714205.8 30453.5 6B 8690.6 15023.6 11633.1 7F 60819.7 53696.7 48511.1 8594.8 58391.9 62852.2 9N 5186.2 208401.8 259323.2 9V 30043.9 38143.948677.2 10A 169.8 36437.6 55124.5 11A 184.5 36771.1 45762.9 12F 130.022278.5 14475.3 14 21906.0 37577.9 64409.8 15B 843.5 22065.2 25248.8 18C91500.7 88824.7 137638.5 19A 16470.7 13070.9 11387.0 19F 13956.4 40516.984553.8 22F 139.7 51649.9 62072.7 23F 12089.4 5772.9 15565.5 33F 143.235602.4 46272.8

When the capsular polysaccharides of serotypes 3 and 5 were conjugatedto TT, the serotype specific IgG concentration significantly increasedcompared to that obtained when they were conjugated to CRM₁₉₇. Rabbitsimmunized with the PCV21(3/5)-TT also demonstrated significant increasesin IgG concentration against the additional eight serotypes not presentin PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). Serotype9N, in particular, when administered with 0.125 mg and 0.250 mg aluminumhad an unexpected 40-fold and 50-fold increase, respectively, in serumspecific IgG concentration relative to PREVNAR13. Further, a number ofthe other capsular polysaccharides of serotypes in PCV21(3/5)-TT showeda significantly higher level of serum specific IgG concentration than inPREVNAR13.

Functional Immunogenicity Test (MOPA)

Antibody functions were evaluated by testing serum in a MOPA assay. S.pneumoniae MOPA strain stored at −70° C. or lower was diluted to thecorresponding final dilution fold so that a concentration of each strainwas about 50,000 CFU/mL. An equivalent amount of serum was sampled fromeach subject, pooled by group and 2-fold serially diluted so that 20 μlof serum remained in a U-bottom plate. After diluting the sample, 10 μlof the strain prepared for each serotype was mixed with the dilutedsample, and the mixture was allowed to react at room temperature for 30minutes so that S. pneumoniae and the antibody were well mixed. Amixture of pre-differentiated HL-60 cells and complement was added andreacted in a CO₂ incubator (37° C.) for 45 minutes. The temperature wasreduced to stop phagocytosis and 10 μl of the reaction solution wasspotted onto an agar plate pre-dried for 30 to 60 minutes, and thenallowed to be absorbed onto the plate for 20 minutes until drying. A 25mg/mL TTC stock solution was added to a prepared overlay agar, and anantibody appropriate for the corresponding strain was added thereto. Themixture was thoroughly mixed, and then about 25 mL of the mixture wasadded onto the plate and hardened for about 30 minutes. The completelyhardened plate was incubated in a CO₂ incubator (37° C.) for 12 to 18hours and then colonies were counted. MOPA titer was expressed as adilution rate at which 50% killings were observed. As a comparativeexample, the commercially-available, 13-valent vaccine (PREVNAR13) wassubjected to the same procedure. The results are shown in Tables 4.

TABLE 4 MOPA titers for 21 serotypes at 2 weeks after secondaryimmunization PCV21(3/5)-TT PCV21(3/5)-TT Serotype PREVNAR13 0.125 mg Al0.250 mg Al 1 94 72 244 3 829 740 3515 4 2428 2698 2675 5 1169 4154 27356A 4925 4567 4761 6B 5693 4959 6629 7F 2731 2095 2103 8 Not tested 710731 9N Not tested 4660 1371 9V 271 280 254 10A Not tested 952 935 11ANot tested 743 1053 12F Not tested 846 737 14 1917 2119 2170 15B Nottested 713 849 18C 5347 2620 4546 19A 5760 2464 2253 19F 2059 2071 213722F Not tested 6022 7783 23F 1975 1487 1566 33F Not tested 824 1924

When the serotypes 3 and 5 were conjugated to TT, functional MOPA titerssignificantly increased compared to MOPA titers obtained when they wereconjugated to CRM₁₉₇. Rabbits immunized with PCV21(3/5)-TT alsodemonstrated significant increases in functional MOPA titers againsteach of the additional eight serotypes that are not present in PREVNAR13(i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F).

4-2. PCV21(1/5)-TT

The serotype specific IgG concentration and functional immunogenicitytiter were measured in the same manner as in 4-1, and the results areshown as follows. Three different PCV21(1/5)-TT embodiments were tested:one with 2.2 μg of capsular polysaccharide from serotype 3 (“3-CRM₁₉₇2.2”), one with 4.4 μg of capsular polysaccharide from serotype 3(“3-CRM₁₉₇ 4.4”) and one with 8.8 μg of capsular polysaccharide fromserotype 3 (“3-CRM₁₉₇ 8.8”).

Serotype Specific IgG Concentration Measurement

TABLE 5 IgG concentration (U/mL) for 21 serotypes at 2 weeks aftersecondary immunization PCV21(1/5)- PCV21(1/5)- PCV21(1/5)- TT TT TT3-CRM₁₉₇ 3-CRM₁₉₇ 3-CRM₁₉₇ Serotype PREVNAR13 2.2 4.4 8.8 1 5105.533530.6 96552.2 44714.9 3 6303.0 7827.2 14676.2 13239.6 4 46727.358893.8 54382.8 39691.0 5 6873.9 14918.4 18743.2 11499.5 6A 32561.411493.2 13053.0 12494.6 6B 25398.7 11856.7 10013.2 4455.6 7F 27560.525799.0 70917.5 56448.2 8 521.0 70060.4 86634.8 63337.9 9N 5198.2169584.3 240858.5 187203.6 9V 62169.3 42445.1 64084.4 34876.4 10A 166.425682.8 44186.9 15827.4 11A 195.3 34050.4 42558.8 33264.2 12F 154.427629.4 38459.7 25874.3 14 17765.9 23787.4 29262.4 24116.4 15B 436.022060.6 21777.0 22573.1 18C 103154.7 119672.1 141889.4 75549.1 19A19191.1 4690.5 5516.9 3407.3 19F 16349.2 31789.8 24370.0 18846.2 22F130.0 40963.1 49475.5 36246.2 23F 15166.5 6312.7 7499.9 5431.5 33F 146.139803.9 38378.5 26837.2

When the capsular polysaccharides of serotypes 1 and 5 were conjugatedto TT, the serotype specific IgG concentration significantly increasedcompared to that obtained when they were conjugated to CRM₁₉₇. Rabbitsimmunized with the PCV21(1/5)-TT also demonstrated significant increasesin IgG concentration against the additional eight serotypes not presentin PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). Again,serotype 9N unexpectedly showed a significant increase (about 32- to46-fold increase) relative to PRENAR13. Further, a number of the othercapsular polysaccharides of serotypes in PCV21(1/5)-TT showed asignificantly higher level of serum specific IgG concentration than inPREVNAR13. It was also observed that doubling the amount of serotype 3antigen from 2.2 μg to 4.4 μg unexpectedly increased the serum specificIgG concentration for serotypes 1, 3, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 18C, 19A, 22F, and 23F, with more than a 2-fold increase forserotypes 1 and 7F.

Functional Immunogenicity Test (MOPA)

TABLE 6 MOPA titers for 21 serotypes at 2 weeks after secondaryimmunization PCV21(1/5)- PCV21(1/5)- PCV21(1/5)- TT TT TT 3-CRM₁₉₇3-CRM₁₉₇ 3-CRM₁₉₇ Serotype PREVNAR13 2.2 4.4 8.8 1 99 198 916 241 3 479565 1072 929 4 2560 3201 2571 2012 5 1046 3717 5430 2025 6A 5624 27085249 2415 6B 5451 3903 4143 2452 7F 2355 2521 2576 2125 8 Not tested 525719 630 9N 54 790 1680 885 9V 282 251 307 183 10A Not tested 684 1065760 11A Not tested 887 1751 747 12F Not tested 856 1016 824 14 1052 12211929 1513 15B 59 1042 823 732 18C 6257 4440 4663 2821 19A 2962 828 1362686 19F 968 1971 1835 1453 22F Not tested 3576 5599 7193 23F 1854 7691358 1050 33F Not tested 932 1982 808

When the serotypes 1 and 5 were conjugated to TT, functional MOPA titerssignificantly increased compared to MOPA titers obtained when they wereconjugated to CRM₁₉₇. Rabbits immunized with the PCV21(1/5)-TT alsodemonstrated significant increases in functional MOPA titers againsteach of the additional eight serotypes that are not present in PREVNAR13(i.e., 8, 9N, 10A, 11A, 12F, 151B, 22F, and 33F). Further, several otherserotypes showed a significantly higher level of functional MOPA titersthan in PREVNAR13. It was also observed that doubling the amount ofserotype 3 antigen from 2.2 μg to 4.4 μg unexpectedly increased theserum specific IgG concentration for each serotype except 4, 151B, and19F, with more than a 4.5-fold increase for serotype 1.

4-3. PCV21(1/5)-TT

The experiments of 4-2 were repeated with a PCV21(1/5)-TT embodimenthaving 2.2 g of capsular polysaccharide from serotype 3 (“3-CRM₁₉₇2.2”), a PCV21(1/5)-TT embodiment having 4.4 μg of capsularpolysaccharide from serotype 3 (“3-CRM₁₉₇ 4.4”), and a PCV21(1/5)-TTembodiment having 4.4 μg of capsular polysaccharides from serotypes 3,4, 6B3, 9V, 19A, and 19F (“Multi-CRM₁₉₇ 4.4”). The serotype specific IgGconcentration and functional immunogenicity titer were measured in thesame manner as in 4-1, and the results are shown as follows.

Serotype Specific IgG Concentration Measurement

TABLE 7 IgG concentration (U/mL) for 21 serotypes at 2 weeks aftersecondary immunization PCV21(1/5)- PCV21(1/5)- PCV21(1/5)- TT TT TT3-CRM₁₉₇ 3-CRM₁₉₇ Multi-CRM₁₉₇ Serotype PREVNAR13 2.2 4.4 4.4 1 14208.334503.1 49226.1 40684.4 3 6575.6 6634.1 13769.4 10841.3 4 16600.333352.0 43605.9 34387.8 5 5079.3 10847.5 20866.2 13405.1 6A 8965.52607.1 13781.5 9194.9 6B 5105.2 1233.5 9225.5 2428.9 7F 59993.0 27961.757039.5 33913.6 8 274.7 56735.3 72822.9 65528.5 9N 3824.7 140424.7181325.9 142736.9 9V 39503.5 29139.3 61759.9 48711.5 10A 130.0 8017.014061.8 15832.8 11A 163.9 33051.7 51222.1 39311.8 12F 130.0 18160.922634.9 14877.6 14 12312.4 12868.7 15953.4 8538.5 15B 280.6 14339.722347.7 22692.0 18C 62963.5 56059.2 141911.5 70910.8 19A 9807.5 1185.74233.4 2649.2 19F 9838.7 9825.8 21175.9 10889.9 22F 130.0 20120.037833.7 21807.5 23F 5835.1 2084.1 4016.2 3152.3 33F 141.4 22732.619748.9 19179.6

As in 4-2, when the capsular polysaccharides of serotypes 1 and 5 wereconjugated to TT, the serotype specific IgG concentration significantlyincreased compared to that obtained when they were conjugated to CRM₁₉₇.Rabbits immunized with the PCV21(1/5)-TT also demonstrated significantincreases in IgG concentration against the additional eight serotypesnot present in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 121, 15B, 22F, and33F), and the capsular polysaccharide of serotype 4 showed asignificantly higher level of serum specific IgG concentration than inPREVNAR13. It was also observed that doubling the amount of serotype 3antigen from 2.2 μg to 4.4 μg unexpectedly increased the serum specificIgG concentration for all but three serotypes. It was also possible touse a higher dose of multiple serotypes, as demonstrated by thePCV21(1/5)-TT embodiment in which the amount of serotypes 3, 4, 6B3, 9V,19A, and 19F was increased from 2.2 μg to 4.4 μg (“Multi-CRM₁₉₇ 4.4”).

Functional Immunogenicity Test (MOPA)

TABLE 8 MOPA titers for 21 serotypes at 2 weeks after secondaryimmunization PCV21(1/5- PCV21(1/5- PCV21(1/5- TT) TT) TT) 3-CRM₁₉₇3-CRM₁₉₇ Multi-CRM₁₉₇ Serotype PREVNAR13 2.2 4.4 4.4 1 109 236 276 255 3740 514 836 768 4 2272 2522 2216 2376 5 3638 12393 18293 2559 6A 49492187 3607 2401 6B 4915 767 5196 1555 7F 2414 2014 2227 1781 8 Not tested582 694 690 9N 84 937 1252 983 9V 295 474 419 290 10A Not tested 566 737674 11A Not tested 801 1666 1878 12F Not tested 600 1008 1298 14 16591503 1200 959 15B 79 902 1459 1794 18C 2933 2594 4095 2968 19A 3910 9571244 751 19F 1570 908 2175 1453 22F Not tested 2268 7833 3516 23F 1956677 786 709 33F Not tested 1021 1115 736

As in 4-2, when the serotypes 1 and 5 were conjugated to TT, thefunctional immunogenicity improved compared to that obtained when theywere conjugated to CRM₁₉₇. Rabbits immunized with the PCV21(1/5)-TT alsodemonstrated significant increases in functional MOPA titers againsteach of the additional eight serotypes conjugated to CRM₁₉₇ that are notpresent in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). Itwas also possible to use a higher dose of multiple serotypes, asdemonstrated by PCV21(1/5)-TT Multi-CRM₁₉₇ 4.4, in which the amount ofserotypes 3, 4, 6B, 9V, 19A, and 19F was increased from 2.2 μg to 4.4μg.

4-4. PCV21(1/5)-TT

The experiments of 4-2 were repeated with a PCV21(1/5)-TT embodimenthaving 4.4 μg of capsular polysaccharide from serotypes 3 and 4(“3,4-CRM₁₉₇ 4.4”). The serotype specific IgG concentration andfunctional immunogenicity titer were measured in the same manner as in4-1, and the results are shown as follows.

Serotype Specific IgG Concentration Measurement

TABLE 9 IgG concentration (U/mL) for 21 serotypes at 2 weeks aftersecondary immunization for 3,4-CRM₁₉₇ 4.4 PCV21 (1,5-TT) SerotypePrevnar13 3,4-CRM₁₉₇ 4.4 1 2512.6 11687.9 3 6160.3 16537.5 4 13018.123834.9 5 7738.6 35815.1 6A 13701.1 6476.3 6B 2290.0 881.1 7F 22885.125523.5 8 170.1 19027.5 9N 971.4 35064.1 9V 13803.2 18139.3 10A 130.07028.4 11A 165.6 10215.7 12F 130.0 3770.0 14 3333.0 4977.4 15B 203.28956.5 18C 28120.5 21748.1 19A 18587.6 6785.8 19F 29732.6 34497.2 22F130.0 21193.6 23F 3962.9 1791.5 33F 130.0 13194.5

When the capsular polysaccharides of serotypes 1 and 5 were conjugatedto TT, the serotype specific IgG concentration significantly increasedcompared to that obtained when they were conjugated to CRM₁₉₇. Rabbitsimmunized with the PCV21(1/5)-TT also demonstrated significant increasesin IgG concentration against the additional eight serotypes not presentin PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). It wasalso observed that doubling the amount of serotype 3 and serotype 4antigen from 2.2 μg to 4.4 μg showed a significantly higher level ofserum specific IgG concentration than in PREVNAR13. It was also possibleto use a higher dose of multiple serotypes, as demonstrated by thePCV21(1/5)-TT embodiment in which the amount of serotypes 3 and 4 wasincreased from 2.2 μg to 4.4 μg (“3,4-CRM₁₉₇ 4.4”).

Functional Immunogenicity Test (MOPA)

TABLE 10 MOPA titers for 21 serotypes at 2 weeks after secondaryimmunization PCV21 (1,5-TT) Serotype Prevnar13 3,4-CRM₁₉₇ 4.4 1 54 455 3393 968 4 2072 3331 5 306 1621 6A 2355 1315 6B 1614 1410 7F 952 981 8 5986 9N 52 2072 9V 324 479 10A 2 427 11A 4 1103 12F 2 266 14 539 969 15B5 255 18C 1996 1392 19A 1870 774 19F 1516 1352 22F 2 1796 23F 928 49633F 2 292

When the serotypes 1 and 5 were conjugated to TT, the functionalimmunogenicity improved compared to that obtained when they wereconjugated to CRM₁₉₇. Rabbits immunized with the PCV21(1/5)-TT alsodemonstrated significant increases in functional MOPA titers againsteach of the additional eight serotypes conjugated to CRM₁₉₇ that are notpresent in PREVNAR13 (i.e., 8, 9N, 10A, 11A, 12F, 15B, 22F, and 33F). Itwas also possible to use a higher dose of multiple serotypes, asdemonstrated by PCV21(1/5)-TT 3,4-CRM₁₉₇ 4.4, in which the amount ofserotypes 3 and 4 was increased from 2.2 μg to 4.4 μg.

Example 5. Additional Details about Preparation of Saccharide-ProteinConjugate from Streptococcus pneumoniae Serotype 9N

Preparation of Cell Bank

Streptococcus pneumoniae serotype 9N (ATCC 6309) was acquired from theAmerican Type Culture Collection (ATCC). For proliferation of the strainand removal of constituents of animal origin, the seed stock wascultured for several generations. The stock vial was kept in arefrigerator (<−70° C.) together with synthetic glycerol as acryoprotectant. For preparation of a cell bank, the cell culture wasproliferated in a soy-based medium. Prior to freezing, the cells wereconcentrated by centrifugation and, after removing the medium used, thecell pellets were resuspended in a fresh medium containing acryoprotectant (e.g., synthetic glycerol).

Fermentation

The culture from the cell bank was inoculated into a seed bottlecontaining a soy-based medium. Until the growth condition was satisfied,the culture was incubated at constant temperature without agitation. Theculture was inoculated into a seed fermenter containing a soy-basedmedium, with temperature, pH and agitation speed controlled, using aseed bottle. Fermentation was terminated after the growth was stopped orthe working capacity of the fermenter was reached. After terminating thefermentation by adding a deactivator, cell debris were removed using acombination of continuous flow centrifugation and filtration.

Purification

The pneumococcal polysaccharide purification process consisted ofmultilayer filtration, repeated concentration/diafiltration andfiltration/elution.

Activation

The final polysaccharide concentration was adjusted to about 2.0 g/L bysequentially adding WFI of a calculated amount. If necessary, thereaction pH was adjusted to approximately 6.0. After the pH adjustment,the reaction temperature was adjusted to 21-25° C. Approximately0.024-0.189 mg of sodium periodate was added per 1 mg of sugar toinitiate oxidation. The oxidation reaction was conducted for 16-20 hoursat 21-25° C.

The activated polysaccharide was concentrated and diafiltered using a100-kDa MWCO ultrafiltration membrane. The diafiltration was conductedfor WFI of 10 times the volume of the diafiltration volume. Then, thepurified activated polysaccharide was stored at 2-8° C. The purifiedactivated saccharide was characterized by (i) the saccharideconcentration determined by colorimetric assay, (ii) the aldehydeconcentration determined by colorimetric assay, (iii) the degree ofoxidation and (iv) the molecular weight measured by SEC-MALLS.

SEC-MALLS is used to determine the molecular weight of polysaccharidesand polysaccharide-protein conjugates. SEC is used to separate thepolysaccharide based on the hydrodynamic volume. A refractive index (RI)detector and a multi-angle laser light scattering (MALLS) detector areused to determine the molecular weight. When light reacts with amaterial, the light is scattered. The quantity of the scattered light isrelated with the concentration, the square of dn/dc (specific refractiveindex increment) and the molar mass of the material. The molecularweight is calculated based on the signal of the scattered light from theMALLS detector and the concentration signal from the RI detector.

The degree of oxidation (DO) of the activated polysaccharide isdetermined as the mole of sugar repeat units divided by the mole ofaldehyde. The mole of sugar repeat units is determined by variouscolorimetric techniques, e.g., using anthrone assay. And, the mole ofaldehyde is determined by the Park-Johnson colorimetric assay.

Using these techniques described above, it was determined that theactivated Streptococcus pneumoniae serotype 9N capsular polysaccharideobtained by the method described above has a degree of oxidation of2-19, more typically 5-10, and a molecular weight of about 200-700 kDa.

Conjugation

The activated polysaccharide was blended with the carrier proteinCRM₁₉₇, at a ratio of 0.5-2 g of CRM₁₉₇ per 1 g of the activatedpolysaccharide. Then, the blended mixture was lyophilized. Thelyophilized mixture of the activated polysaccharide and CRM₁₉₇ wasstored at −20° C.

The lyophilized mixture of the activated polysaccharide and CRM₁₉₇ wasreconstituted in a 0.1 M sodium phosphate solution and then mixedsufficiently. The final polysaccharide concentration in the reactionsolution was about 10-20 g/L. After initiating conjugation by adding1.0-1.2 molar equivalents of sodium cyanoborohydride (NaBH₃CN) to thereaction mixture, the reaction was conducted at 35-39° C. for 44-52hours. The conjugation reaction was terminated by adding a 0.9% sodiumchloride solution of the same volume as the conjugation reactionsolution and then adding 1.8-2.2 molar equivalents of sodium borohydride(NaBH₄) to cap the unreacted aldehyde. The capping reaction wasconducted at 21-25° C. for 3-6 hours.

The conjugate solution was diluted with a 0.9% sodium chloride solutionfor concentration and diafiltration using a 100-kDa MWCO membrane. Thediluted conjugate solution was filtered through a 0.8-0.45 μm filter andpurified by concentration and diafiltration. The diafiltration using a100-kDa MWCO membrane was conducted using a 0.9% sodium chloridesolution of 15-40 times the diafiltration volume. After thediafiltration was completed, the remaining solution was filtered througha 0.2 μm filter. The conjugate solution was diluted to a concentrationbelow approximately 0.55 mg/mL, sterile-filtered and then stored at 2-8°C.

The purified serotype 9N conjugate was characterized, in particular, by(i) the protein concentration determined by colorimetric (Lowry) assay,(ii) the aldehyde concentration determined by colorimetric assay, (iii)the saccharide-to-protein ratio, (iv) the molecular size distributiondetermined by size exclusion chromatography (CL-4B) and (v) themolecular weight measured by SEC-MALLS.

The change in the characteristics of the serotype 9N conjugate wasobserved while varying the degree of oxidation (DO). The result issummarized in Table 11.

TABLE 11 Conjugate number 1 2 3 4 5 6 Molecular weight of activated 582619 459 563 490 427 polysaccharide, kDa DO 18.2 9.4 7.4 6.7 4.3 2.3Input ratio (P:S) 0.8:1 Polysaccharide concentration 20.0 in conjugationreaction solution, g/L % conjugate yield 53 43 39 32 33 39Saccharide-to-protein ratio 2.1 1.5 1.3 1.1 1.0 0.78 % free saccharide44 28 22 20 21 31 % molecular weight 52 49 50 55 44 31 distributionMolecular weight of conjugate, 860 1,110 1,912 1,168 1,189 1,160 kDa

The change in the characteristics of the serotype 9N conjugate wasobserved while varying the blending ratio of the activatedpolysaccharide and CRM₁₉₇ during the lyophilization. The result issummarized in Table 12.

TABLE 12 Conjugate number 7 8 9 10 11 Molecular weight of activated 287polysaccharide, kDa DO 5.6 Input ratio (P:S) 2:1 1.5:1 1:1 0.67:1 0.5:1Polysaccharide concentration in 20.0 conjugation reaction solution, g/L% conjugate yield 25 50 43 41 66 Saccharide-to-protein ratio 0.71 0.851.0 1.2 1.8 % free saccharide 5 6 15 27 62 % molecular weightdistribution 52 58 50 40 22 Molecular weight of conjugate, kDa 3,7203,713 1,327 1,016 545

The change in the characteristics of the serotype 9N conjugate wasobserved while varying the polysaccharide concentration in theconjugation reaction solution. The result is summarized in Table 13.

TABLE 13 Conjugate number 12 13 14 15 16 Molecular weight of activated560 polysaccharide, kDa DO 6.1 Input ratio (P:S) 0.8:1 Polysaccharideconcentration in 10.0 12.5 15.0 17.5 20.0 conjugation reaction solution,g/L % conjugate yield 20 31 28 40 42 Saccharide-to-protein ratio 1.0 1.00.93 0.99 0.97 % free saccharide 32 30 22 21 18 % molecular weightdistribution 17 27 40 47 54 Molecular weight of conjugate, kDa 560 546845 932 1,438

Example 6. Analysis of Immunogenicity

A monovalent conjugate composition containing the Streptococcuspneumoniae serotype 9N saccharide-protein conjugate conjugated to CRM₁₉₇was formulated.

The immunogenicity of the monovalent immunogenic compositions of Tables11-13 was analyzed by ELISA. The serum concentration of theserotype-specific IgG was determined.

Five female New Zealand white rabbits weighing 2.5-3.5 kg were immunizedwith the proposed human clinical dose (conjugate 2.2 μg; +0.25 mg/mLaluminum as AlPO₄) at 0 week via an intramuscular route. The rabbitswere immunized again at week 2 with the conjugate vaccine of the samedose and blood samples were taken at week 4. Serotype-specific ELISA wasconducted for serum samples at week 0 and week 4.

The analysis result is shown in Table 14. The rabbits immunized with themonovalent conjugate composition (conjugate number 8) showed significantincrease in the total IgG titer for the serotype 9N. The rabbitsimmunized with other conjugates also showed significant increase in thetotal IgG titer.

Table 14 shows a result of measuring the IgG concentration afterimmunizing the rabbits with the conjugate number 8 of Table 12.

TABLE 14 IgG concentration (U/mL) Serotype Pre-immunizationPost-immunization 9N 130.0 656,345.3

While one or more exemplary embodiments have been described in thespecification, it will be understood by those of ordinary skill in theart that various changes in form and details may be made therein withoutdeparting from the spirit and scope of the inventive concept as definedby the following claims.

REFERENCES

The following references are cited in the application and providegeneral information regarding the technical field and provide assays andother details discussed in the application. The following references areincorporated herein by reference in their entirety.

-   [1] Prymula et al., The Lancet, 367:740-48 (2006).-   [2] Vesikari et al., PIDJ, 28(4):S66-76 (2009).-   [3] Dagan et al., Infection & Immunity, 5383-91 (2004).-   [4] Juergens et al., Clinical and Vaccine Immunology,    21(9):1277-1281 (2014).-   [5] Andrews et al., The Lancet, 14:839-846 (2014).-   [6] Nurkka et al., Vaccine, 20:194-201 (2001).-   [7] Levin and Stone, J. Immunol., 67:235-242 (1951).-   [8] W.H.O. Manual for the Production and Control of Vaccines:    Tetanus Toxoid, 1977 (BLG/UNDP/77.2 Rev.I.)-   [9] Didierlaurent et al., J. Immunol., 183:6186-6197 (2009).

1-10. (canceled)
 11. A vaccine comprising a pharmaceutically acceptableexcipient and a mixed carrier, multivalent pneumococcal conjugatecomposition comprising 21 different pneumococcal capsularpolysaccharide-protein conjugates, wherein each pneumococcal capsularpolysaccharide-protein conjugate comprises a protein carrier conjugatedto a capsular polysaccharide from a different serotype of Streptococcuspneumoniae, wherein the Streptococcus pneumoniae serotypes are selectedfrom 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F, and 33F, wherein the protein carrier is CRM₁₉₇ ortetanus toxoid, and wherein two of the capsular polysaccharides areconjugated to tetanus toxoid and the remaining capsular polysaccharidesare conjugated to CRM₁₉₇, wherein the two capsular polysaccharides thatare conjugated to tetanus toxoid are from serotypes 1 and 3 or serotypes3 and
 5. 12-21. (canceled)
 22. The vaccine of claim 11, wherein thecapsular polysaccharides from serotypes 1 and 3 are conjugated to thetetanus toxoid, and the capsular polysaccharides from serotypes 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B 18C, 19A, 19F, 22F, 23F,and 33F are conjugated to CRM₁₉₇.
 23. The vaccine of claim 11, whereinthe capsular polysaccharides from serotypes 3 and 5 are conjugated tothe tetanus toxoid, and the capsular polysaccharides from serotypes 1,4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B 18C, 19A, 19F, 22F,23F, and 33F are conjugated to CRM₁₉₇.
 24. The vaccine of claim 22,wherein the vaccine is formulated as a single 0.5 ml dose comprising 2μg to 2.5 μg of each capsular polysaccharide except the capsularpolysaccharide of serotype 6B, and 4 μg to 5 μg of capsularpolysaccharide from serotype 6B.
 25. The vaccine of claim 24, whereinthe vaccine is formulated as a single 0.5 ml dose comprising 2.2 μg ofeach capsular polysaccharide except the capsular polysaccharide ofserotype 6B, and 4.4 μg of capsular polysaccharide from serotype 6B. 26.The vaccine of claim 22, wherein the vaccine is formulated as a single0.5 ml dose comprising 2 μg to 2.5 μg of each capsular polysaccharidefrom serotypes 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F,23F, and 33F and 4 μg to 5 μg of each capsular polysaccharide fromserotypes 3, 4, 6B, 9V, 19A, and 19F.
 27. The vaccine of claim 22,wherein the vaccine is formulated as a single 0.5 ml dose comprising 2.2μg of each capsular polysaccharide from serotypes 1, 5, 6A, 7F, 8, 9N,10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F and 4.4 μg of eachcapsular polysaccharide from serotypes 3, 4, 6B, 9V, 19A, and 19F. 28.The vaccine of claim 22, wherein the vaccine is formulated as a single0.5 ml dose comprising 2.2 μg of each capsular polysaccharide except forup to six serotypes selected from the group consisting of serotypes 1,3, 4, 5, 6B, 9V, 19A, and 19F, and 4.4 μg of each capsularpolysaccharide from the up to six serotypes selected from the groupconsisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F.
 29. Thevaccine of claim 22, wherein the vaccine is formulated as a single 0.5ml dose comprising: 2 μg to 2.5 μg of each capsular polysaccharideexcept for serotype 6B; 4 μg to 5 μg of capsular polysaccharide fromserotype 6B; 2 μg to 25 μg of tetanus toxoid; 40 μg to 75 μg of CRM₁₉₇;and 0.1 to 0.250 mg of elemental aluminum adjuvant.
 30. The vaccine ofclaim 29, wherein the vaccine comprises: 2.2 μg of each capsularpolysaccharide except for serotype 6B; 4.4 μg of capsular polysaccharidefrom serotype 6B; 2 μg to 25 μg of tetanus toxoid; 40 μg to 75 μg ofCRM₁₉₇; 0.125 to 0.250 mg of elemental aluminum adjuvant; sodiumchloride, and a succinate buffer.
 31. The vaccine of claim 30, furthercomprising polysorbate
 80. 32. The vaccine of claim 30, wherein theelemental aluminum adjuvant comprises aluminum phosphate.
 33. A methodfor prophylaxis of Streptococcus pneumoniae infection or disease causedby Streptococcus pneumoniae infection in a subject, the methodcomprising administering a prophylactically effective amount of thevaccine of claim 22 to the subject.
 34. The method of claim 33, whereinthe subject is a human who is at least 50 years old and the disease ispneumonia or invasive pneumococcal disease (IPD).
 35. The method ofclaim 33, wherein the subject is a human who is at least 6 weeks old andthe disease is pneumonia, invasive pneumococcal disease (IPD), or acuteotitis media (AOM).
 36. The method of claim 35, wherein the subject is 6weeks to 5 years of age, 2 to 15 months of age, or 6 to 17 years of age.37. The method of claim 33, wherein the subject is a human.
 38. Themethod of claim 33, wherein the vaccine is administered by intramuscularinjection.
 39. The method of claim 33, wherein the vaccine isadministered as part of an immunization series.
 40. A method forprophylaxis of Streptococcus pneumoniae infection or disease caused byStreptococcus pneumoniae infection in a human subject, the methodcomprising administering a prophylactically effective amount of thevaccine of claim 30 to the human subject.
 41. The vaccine of claim 23,wherein the vaccine is formulated as a single 0.5 ml dose comprising 2μg to 2.5 μg of each capsular polysaccharide except the capsularpolysaccharide of serotype 6B, and 4 μg to 5 μg of capsularpolysaccharide from serotype 6B.
 42. The vaccine of claim 41, whereinthe vaccine is formulated as a single 0.5 ml dose comprising 2.2 μg ofeach capsular polysaccharide except the capsular polysaccharide ofserotype 6B, and 4.4 μg of capsular polysaccharide from serotype 6B. 43.The vaccine of claim 23, wherein the vaccine is formulated as a single0.5 ml dose comprising 2 μg to 2.5 μg of each capsular polysaccharidefrom serotypes 1, 5, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 18C, 22F,23F, and 33F and 4 μg to 5 μg of each capsular polysaccharide fromserotypes 3, 4, 6B, 9V, 19A, and 19F.
 44. The vaccine of claim 23,wherein the vaccine is formulated as a single 0.5 ml dose comprising 2.2μg of each capsular polysaccharide from serotypes 1, 5, 6A, 7F, 8, 9N,10A, 11A, 12F, 14, 15B, 18C, 22F, 23F, and 33F and 4.4 μg of eachcapsular polysaccharide from serotypes 3, 4, 6B, 9V, 19A, and 19F. 45.The vaccine of claim 23, wherein the vaccine is formulated as a single0.5 ml dose comprising 2.2 μg of each capsular polysaccharide except forup to six serotypes selected from the group consisting of serotypes 1,3, 4, 5, 6B, 9V, 19A, and 19F, and 4.4 μg of each capsularpolysaccharide from the up to six serotypes selected from the groupconsisting of serotypes 1, 3, 4, 5, 6B, 9V, 19A, and 19F.
 46. Thevaccine of claim 23, wherein the vaccine is formulated as a single 0.5ml dose comprising: 2 μg to 2.5 μg of each capsular polysaccharideexcept for serotype 6B; 4 μg to 5 μg of capsular polysaccharide fromserotype 6B; 2 μg to 25 μg of tetanus toxoid; 40 μg to 75 μg of CRM₁₉₇;and 0.1 to 0.250 mg of elemental aluminum adjuvant.
 47. The vaccine ofclaim 46, wherein the vaccine comprises: 2.2 μg of each capsularpolysaccharide except for serotype 6B; 4.4 μg of capsular polysaccharidefrom serotype 6B; 2 μg to 25 μg of tetanus toxoid; 40 μg to 75 μg ofCRM₁₉₇; 0.125 to 0.250 mg of elemental aluminum adjuvant; sodiumchloride, and a succinate buffer.
 48. The vaccine of claim 47, furthercomprising polysorbate
 80. 49. The vaccine of claim 48, wherein theelemental aluminum adjuvant comprises aluminum phosphate.
 50. A methodfor prophylaxis of Streptococcus pneumoniae infection or disease causedby Streptococcus pneumoniae infection in a subject, the methodcomprising administering a prophylactically effective amount of thevaccine of claim 23 to the subject.
 51. The method of claim 50, whereinthe subject is a human who is at least 50 years old and the disease ispneumonia or invasive pneumococcal disease (IPD).
 52. The method ofclaim 50, wherein the subject is a human who is at least 6 weeks old andthe disease is pneumonia, invasive pneumococcal disease (IPD), or acuteotitis media (AOM).
 53. The method of claim 52, wherein the subject is 6weeks to 5 years of age, 2 to 15 months of age, or 6 to 17 years of age.54. The method of claim 50, wherein the subject is a human.
 55. Themethod of claim 50, wherein the vaccine is administered by intramuscularinjection.
 56. The method of claim 50, wherein the vaccine isadministered as part of an immunization series.
 57. A method forprophylaxis of Streptococcus pneumoniae infection or disease caused byStreptococcus pneumoniae infection in a human subject, the methodcomprising administering a prophylactically effective amount of thevaccine of claim 47 to the human subject.